Systems and methods for isolating and using clinically safe adipose derived regenerative cells

ABSTRACT

Systems and methods are described that are used to separate cells from a wide variety of tissues. In particular, automated systems and methods are described that separate regenerative cells, e.g., stem and/or progenitor cells, from adipose tissue. The systems and methods described herein provide rapid and reliable methods of separating and concentrating regenerative cells suitable for re-infusion into a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/554,755, filed on Sep. 4, 2009, and entitled “SYSTEMS AND METHODS FORISOLATING AND USING CLINICALLY SAFE ADIPOSE-DERIVED REGENERATIVE CELLS,”which is a divisional of U.S. application Ser. No. 10/884,638, filed onJul. 2, 2004, entitled “SYSTEMS AND METHODS FOR ISOLATING AND USINGCLINICALLY SAFE ADIPOSE-DERIVED REGENERATIVE CELLS”, now issued U.S.Pat. No. 7,585,670, which is a continuation-in-part application of U.S.application Ser. No. 10/316,127, filed on Dec. 9, 2002, entitled SYSTEMSAND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS,which claims the benefit of U.S. Provisional Application No. 60/338,856,filed Dec. 7, 2001. The contents of all the aforementioned applicationsare expressly incorporated herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to systems and methods for separating andconcentrating cells, e.g., regenerative cells, from a wide variety oftissues. The present invention particularly relates to separating andconcentrating clinically safe regenerative cells from adipose tissueusing the systems and methods of the present invention.

2. Description of the Related Art

Regenerative medicine harnesses, in a clinically targeted manner, theability of regenerative cells, e.g., stem cells and/or progenitor cells(i.e., the unspecialized master cells of the body), to renew themselvesindefinitely and develop into mature specialized cells. Stem cells arefound in embryos during early stages of development, in fetal tissue andin some adult organs and tissue (Pera et al., 2000). Embryonic stemcells (hereinafter referred to as “ESCs”) are known to become many ifnot all of the cell and tissue types of the body. ESCs not only containall the genetic information of the individual but also contain thenascent capacity to become any of the 200+ cells and tissues of thebody. Thus, these cells have tremendous potential for regenerativemedicine. For example, ESCs can be grown into specific tissues such asheart, lung or kidney which could then be used to repair damaged anddiseased organs (Assady et al., 2001; Jacobson et al., 2001; Odorico etal., 2001). However, ESC derived tissues have clinical limitations.Since ESCs are necessarily derived from another individual, i.e., anembryo, there is a risk that the recipient's immune system will rejectthe new biological material. Although immunosuppressive drugs to preventsuch rejection are available, such drugs are also known to blockdesirable immune responses such as those against bacterial infectionsand viruses. Moreover, the ethical debate over the source of ESCs, i.e.,embryos, is well-chronicled and presents an additional and, perhaps,insurmountable obstacle for the foreseeable future.

Adult stem cells (hereinafter interchangeably referred to as “ASCs”)represent an alternative to the use of ESCs. ASCs reside quietly in manynon-embryonic tissues, presumably waiting to respond to trauma or otherdestructive disease processes so that they can heal the injured tissue(Arvidsson et al., 2002; Bonner-Weir and Sharma, 2002; Clarke andFrisen, 2001; Crosby and Strain, 2001; Jiang et al., 2002a). Notably,emerging scientific evidence indicates that each individual carries apool of ASCs that may share with ESCs the ability to become many if notall types of cells and tissues (Young et al., 2001; Jiang et al., 2002a;Jiang et al., 2002b; Schwartz et al., 2002). Thus, ASCs, like ESCs, havetremendous potential for clinical applications of regenerative medicine.

ASC populations have been shown to be present in one or more of bonemarrow, skin, muscle, liver and brain (Jiang et al., 2002b; Alison,1998; Crosby and Strain, 2001). However, the frequency of ASCs in thesetissues is low. For example, mesenchymal stem cell frequency in bonemarrow is estimated at between 1 in 100,000 and 1 in 1,000,000 nucleatedcells (D'Ippolito et al., 1999; Banfi et al., 2001; Falla et al., 1993).Similarly, extraction of ASCs from skin involves a complicated series ofcell culture steps over several weeks (Toma et al., 2001) and clinicalapplication of skeletal muscle-derived ASCs requires a two to three weekculture phase (Hagege et al., 2003). Thus, any proposed clinicalapplication of ASCs from such tissues requires increasing cell number,purity, and maturity by processes of cell purification and cell culture.

Although cell culture steps may provide increased cell number, purity,and maturity, they do so at a cost. This cost can include one or more ofthe following technical difficulties: loss of cell function due to cellaging, loss of potentially useful non-stem cell populations, delays inpotential application of cells to patients, increased monetary cost, andincreased risk of contamination of cells with environmentalmicroorganisms during culture. Recent studies examining the therapeuticeffects of bone-marrow derived ASCs have used essentially whole marrowto circumvent the problems associated with cell culturing (Horwitz etal., 2001; Orlic et al., 2001; Stamm et al., 2003; Strauer et al.,2002). The clinical benefits, however, have been suboptimal, an outcomealmost certainly related to the limited ASC dose and purity inherentlyavailable in bone marrow.

Recently, adipose tissue has been shown to be a source of ASCs (Zuk etal., 2001; Zuk et al., 2002). Unlike marrow, skin, muscle, liver andbrain, adipose tissue is comparably easy to harvest in relatively largeamounts (Commons et al., 2001; Katz et al., 2001b). Furthermore, adiposederived ASCs have been shown to possess the ability to generate multipletissues in vitro, including bone, fat, cartilage, and muscle (Ashjian etal., 2003; Mizuno et al., 2002; Zuk et al., 2001; Zuk et al., 2002).Thus, adipose tissue presents an optimal source for ASCs for use inregenerative medicine.

Suitable methods for harvesting adipose derived ASCs, however, may belacking in the art. Existing methods may suffer from a number ofshortcomings. For example, the existing methods may lack the ability tooptimally accommodate an aspiration device for removal of adiposetissue. The existing methods may also lack partial or full automationfrom the harvesting of adipose tissue phase through the processing oftissue phases (Katz et al., 2001a) and/or. The existing methods furthermay lack volume capacity greater than 100 ml of adipose tissue. Theexisting methods may yet further lack a partially or completely closedsystem from the harvesting of adipose tissue phase through theprocessing of tissue phases. Finally, the existing methods may lackdisposability of components to attenuate concomitant risks ofcross-contamination of material from one sample to another. In summary,the many prior art methods for harvesting ASCs from adipose tissue donot appear to overcome the technical difficulties associated withharvesting ASCs from skin, muscle, liver and brain described above.Accordingly, there remains a need in the art for systems and methodsthat are capable of harvesting regenerative cell populations, e.g.,ASCs, with increased yield, consistency and/or purity and of doing sorapidly and reliably with a diminished or non-existent need forpost-extraction manipulation.

Ideally, such a device, system or method would yield regenerative cellsin a manner suitable for direct placement into a recipient. Towards thisend, the system or method of the present invention is optimized suchthat direct placement or re-infusion of the regenerative cells from thesystem into the patient does not provoke an adverse event in thepatient, e.g., such as those caused by the presence of unsafe levels ofendotoxins, infectious agents, bacteria, and other additives.

SUMMARY OF THE INVENTION

The present invention relates to highly versatile system and methodscapable of separating and concentrating a given tissue to produceclinically safe regenerative cells, e.g., stem and progenitor cells,suitable for re-infusion into a subject. In a preferred embodiment, thepresent invention provides an automated system for separating andconcentrating clinically safe regenerative cells from adipose tissuethat are suitable for re-infusion into a subject. A system forseparating and concentrating cells from adipose tissue in accordancewith the disclosure herein generally includes one or more of acollection chamber, a processing chamber, a waste chamber, an outputchamber and a sample chamber. The various chambers are coupled togethervia one or more conduits such that fluids containing biological materialmay pass from one chamber to another in a closed, or functionallyclosed, sterile fluid/tissue pathway which minimizes exposure of tissue,cells, biologic and non-biologic materials with contaminants. In certainembodiments, the waste chamber, the output chamber and the samplechamber are optional. In a preferred embodiment, the system containsclinically irrelevant quantities of endotoxin.

The system also includes a plurality of filters. The filters areeffective to separate the stem cells and/or progenitor cells from, amongother things, collagen, free lipids, adipocytes, and tissuedisaggregation agents, that may be present in the solution in connectionwith the processing of adipose tissue. In one embodiment, a filterassembly includes a hollow fiber filtration device. In anotherembodiment, a filter assembly includes a percolative filtration device,which may or may not be used with a sedimentation process. In apreferred embodiment, the filter assembly comprises a centrifugationdevice, which may or may not be used with an elutriation device andprocess. In yet another embodiment, the system comprises a combinationof these filtering devices. The filtration functions of the presentinvention can be two-fold, with some filters removing things from thefinal concentration such as collagen, free lipid, free adipocytes andresidual collagenase, and with other filters being used to concentratethe final product.

In other embodiments, one or more components of the system are automatedand include an internal processing device and associated softwareprograms which control many of the processing functions. Components ofthe system may be disposable, such that portions of the system can bedisposed of after a single use. Such a system also comprises a re-usablecomponent which includes the processing device (computer and associatedsoftware programs) and other components such as motors, pumps, etc.

In one embodiment, a method of treating a patient includes steps of: a)providing a tissue removal system; b) removing adipose tissue from apatient using the tissue removal system, the adipose tissue having aconcentration of stem cells; c) processing at least a part of theadipose tissue to obtain a concentration of regenerative cells otherthan the concentration of regenerative cells of the adipose tissuebefore processing, wherein the processing occurs within a sterile,closed or functionally closed system; and d) administering theregenerative cells to a patient without removing the regenerative cellsfrom the tissue removal system before being administered to the patient,to thereby treat the patient.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone skilled in the art.

Additional advantages and aspects of the present invention are apparentin the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is an illustration of a system for separating and concentratingregenerative cells from tissue which includes one filter assembly.

FIG. 2 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a serial configuration.

FIG. 3 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a parallel configuration.

FIG. 4 is an illustration of a system for separating and concentratingregenerative cells from tissue which includes a centrifuge chamber.

FIG. 5 is a sectional view of a collection chamber including a prefixedfilter utilized in a system for separating and concentratingregenerative cells from tissue.

FIG. 6 is a sectional view of a processing chamber of a system forseparating and concentrating regenerative cells from tissue utilizing apercolative filtration system.

FIG. 7 is a sectional view of a processing chamber of a system forseparating and concentrating regenerative cells utilizing a centrifugedevice for concentrating the regenerative cells.

FIG. 8 is another sectional view of the processing chamber of FIG. 7.

FIGS. 9.1, 9.2 and 9.3 illustrate an elutriation component in use withthe system of the invention.

FIG. 10 is an illustration of a system for separating and concentratingregenerative cells from tissue utilizing vacuum pressure to move fluidsthrough the system. A vacuum system can be constructed by applying avacuum pump or vacuum source to the outlet of the system, controlled ata predetermined rate to pull tissue and fluid through, using a system ofstopcocks, vents, and clamps to control the direction and timing of theflow.

FIG. 11 is an illustration of a system for separating and concentratingregenerative cells from tissue utilizing positive pressure to movefluids through the system. A positive pressure system uses a mechanicalmeans such as a peristaltic pump to push or propel the fluid and tissuethrough the system at a determined rate, using valves, stopcocks, vents,and clamps to control the direction and timing of the flow.

FIG. 12A illustrates a filtration process in which the feed stream offluid flows tangentially to the pores of the filter. FIG. 12Billustrates a filtration process in which the feed stream of fluid flowsperpendicular to the pores of the filter.

FIG. 13 is an illustration of an exemplary disposable set for a systemof the invention.

FIG. 14 is an illustration of an exemplary re-usable component for asystem of the invention.

FIG. 15A is an illustration of an exemplary device of the inventionassembled using the disposable set of FIG. 13 and a re-usable componentof FIG. 14.

FIG. 15B is a flowchart depicting exemplary pre-programmed steps,implemented through a software program, that control automatedembodiments of a system of the present invention. Two alternativeprocessing parameters are shown indicating the versatility of thesystem.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to rapid and reliable systems and methodsfor separating and concentrating clinically safe regenerative cells,e.g., stem cells and/or progenitor cells, from a wide variety oftissues, including but not limited to, adipose, bone marrow, blood,skin, muscle, liver, connective tissue, fascia, brain and other nervoussystem tissues, blood vessels, and other soft or liquid tissues ortissue components or tissue mixtures (e.g., a mixture of tissuesincluding skin, blood vessels, adipose, and connective tissue). In apreferred embodiment, the system separates and concentrates clinicallysafe regenerative cells from adipose tissue. In a particularly preferredembodiment, the clinically safe regenerative cells obtained using thesystems and methods of the present invention are suitable for placementinto a recipient.

The clinically safe regenerative cells of the invention arecharacterized by both, the absence of contaminants e.g., endotoxin,residual enzymes, free lipid, and, in certain embodiments, collagenfragments, as well as by the presence of regenerative cell populations,e.g., stem cells, progenitor cells, endothelial cells, etc. Accordingly,the systems and methods of the present invention are optimized such thatre-infusing a patient with the clinically safe regenerative cellsobtained using the systems and methods of the present invention providesa therapeutic benefit (i.e., the regenerative cells are viable andretain regenerative function) that outweighs the potential for risk ofadverse events.

For example, the biologic, non-biologic and other components of thesystems and methods of the present invention do not contribute toclinically relevant levels of endotoxins present in the regenerativecells or in any intermediate regenerative cell compositions.Accordingly, the regenerative cells obtained do not contain endotoxin atlevels that might elicit an adverse event when placed within a patient.In addition, the properties of endotoxin-sensitive cells within theregenerative cells obtained are not altered such that infusion of saidcells into a patient could result in an adverse event that would nothave occurred in the absence of endotoxin exposure.

In addition, the biologic, non-biologic and other components of thesystems and methods of the present invention do not add bacteria orother infectious agents to the regenerative cells or to any regenerativecell composition intermediates. Accordingly, the regenerative cellsobtained do not elicit an adverse event when placed within a recipient.Also, the properties of sensitive cells within the regenerative cellsobtained are not altered such that infusion of said cells results in anadverse event that would not have occurred in the absence of exposure tosaid agent

Similarly, the biologic, non-biologic and other components of thesystems and methods of the present invention do not contaminate to theregenerative cells or to any regenerative cell composition intermediateswith cells, proteins, fluids, or other material coming from anindividual other than the person into whom the regenerative cells are tobe placed. The term “contaminant” does not include material added topromote efficient processing or delivery of the cells (for example,human serum albumin which might be added prior to intravascularadministration). Such additives would not be considered contaminants asthey are added intentionally and as used herein, “contaminant” refers toany protein, cell, fluid, agent or other material which inadvertently(in the absence of design or knowledge of the operator) becomes includedinto the regenerative cells obtained using the systems and methods ofthe present invention or which changes the properties of theregenerative cells obtained in a manner that would not have occurred inthe absence of said material.

Furthermore, any additives capable of causing an adverse reaction in apatient which may be present or added to the biologic, non-biologic andother components of the systems and methods of the present invention areremoved from the regenerative cells. For example, proteolytic enzymesadded to degrade extracellular matrix within the adipose tissue.Placement of such enzymes within the tissues of a recipient could leadto degradation of extracellular matrix material within the recipient andsubsequent adverse events. In addition, degraded or partially degradedextracellular matrix proteins with the ability to induce plateletaggregation and subsequent thrombotic events or to elicit an autoimmuneresponse are removed. Also free lipids are removed such that quantitiessufficient to create a substantial risk of embolism are not present.

The system includes one or several automated sampling probes placed inline with digested adipose tissue at various stages in the processing,including, but not limited to the final regenerative cell suspension, inorder to sample the digested adipose tissue materials for potentiallyunsafe contaminants. Such a probe may be used to test the supernatant orcell suspension of the final regenerative cell preparation. Such a probeis designed to either dispense a cell suspension directly into a testingchamber or into a cell concentration device, such as a centrifuge orelutriator, in order to isolate the supernatant of the cell suspension,which is subsequently dispensed into a testing chamber.

A sampling probe described above for sampling the final regenerativecell suspension may be used with a testing chamber for adipocytes. Thistesting chamber may consist of the following components; (1) a stage forholding a microscope slide, and (2) an automated cover slipping unit.The sampling probe is positioned in such as way as to obtain a sample ofthe final regenerative cell suspension from the main compartment andthen to deliver the sample into a component of the testing chamber. Theoperation of such a testing chamber would proceed as follows: (1)) theoperator has pre-placed a miscroscope slide onto the stage, (2) a sampleof the cell suspension is obtained by the sampling probe, (2) the probethen moves in an automated fashion above the microscope slide, (3) thestage is then automatically set to move slowly in a lateral plane while(4) the sampling probe dispenses a thin layer of the cell suspensionacross the microscope slide, and (5) the automated cover slipping unitplaces a cover slip atop of the slide after the sample has beendispensed onto the slide. The slide is then held in place until theoperator removes it from the testing chamber, stains it with Oil Red Oor some other adipocyte selective antibody based or non-antibody basedstain, and quantifies the cells microscopically.

A sampling probe described above for sampling the final regenerativecell suspension may also be used with a testing chamber to test for freelipid in the cell suspension. This testing chamber may consist of thefollowing components; (1) a conduit that is a port which connects theoutside of the entire device with the testing chamber (2) aspectrophotometer or fluorimeter that houses a unit that holds tube(s)or well(s) for placement of the cell sample and a triglyceride reactivechromagen or fluorogen and that is connected to a digital display on theoutside of the entire device that converts the chromogenic orfluorometric signal into triglyceride content, a measure of free lipid.The operation of such a testing chamber would proceed as follows: (1)The probe samples the cell suspension and (2) dispenses it into the tubeor well (3) The operator injects an appropriate amount of the chromagenor fluorogen into the port, such that the solution is dispensed intotube(s) or well(s), (4) the tube or well and its contents incubate foran appropriate, designated period of time under controlled temperature,(5) the spectrophotometer or fluorimeter reads the contents of the tubeor well, and (6) the lipid content in the sample is displayed digitallyon the outside of the device.

A sampling probe in line with the final regenerative cell suspension mayalso be used with a testing chamber to test for residual, solubleproteolytic activity in the supernatant of the cell suspension. Such achamber would consist of the following major components; (1) acentrifuge that separates out the cell pellet from the supernatant, 2) aspectrophotometer or fluorimeter that houses a unit that holds tube(s)or well(s) for placement of the regenerative cell sample and acolorigenic or fluorigenic protease substrate, and is connected to adigital display on the outside of the entire device that converts thechromogenic or fluorometric signal into proteolytic activity, such ascollagenase or thermolysin activity as measured by gelatin or caseindigestion, respectively. The operation of such a testing chamber wouldgo as follows: (1) The probe samples the final regenerative cellsuspension and (2) dispenses it into a chamber within the centrifugewhich then automatically begins revolving at a predefined g force andtime to separate out the cell pellet and supernatant, (3) the samplingprobe then obtains a sample of the supernatant from the finalregenerative cell suspension (4) the operator injects an appropriateamount of the chromagenic or fluorogenic protease substrate into theport, such that the solution is dispensed into the spectrophotometer orfluorimeter tube(s) or well(s), (5) the tube(s) or well(s) and itscontents incubate for an appropriate, designated period of time undercontrolled temperature, (6) the spectrophotometer or fluorimeter readsthe contents of the tube(s) or well(s), and (7) the proteolytic activityin the sample is displayed digitally on the outside of the device.

A sampling probe in line with the final regenerative cell suspension mayalso be used with a testing chamber to test for soluble factors from thesupernatant of the final regenerative cell suspension, or cells from thefinal regenerative cell suspension, that can induce plateletaggregation. Such a chamber would consist of the following majorcomponents; (1) a centrifuge that separates out the cell pellet from thesupernatant, (2) a temperature controlled aggregometer that contains aunit for holding tube(s) or well(s) and is connected to a digitaldisplay on the outside of the entire device that converts the amount ofturbidity associated with platelet aggregation into a unit of plateletaggregation that is then displayed digitally on the outside of theentire device, and that has two separate ports; (a) one port thatdelivers the supernatant of the final regenerative cell preparation intothe testing chamber and (b) one port that connects the outside of theentire device to the testing chamber. The operation of such a testingchamber would go as follows: (1) The operator injects platelet richplasma (PRP) into the port connected between the chamber and the outsideof the device such that the PRP is dispensed into the tube(s) or well(s)within the aggregometer, (2) the sampling probe obtains a sample of thefinal regenerative cell suspension and performs the step “3” ifmeasuring soluble agonists of platelet aggregation or performs step “4”if measuring cell agonists of platelet aggregation, (3) the samplingprobe dispenses the sample into a chamber within the centrifuge whichthen automatically begins revolving at a predefined g force and time toseparate out the cell pellet and supernatant, then the sampling probeobtains a sample of the supernatant and dispenses into the tube(s) orwell(s) within the aggregometer (4) the sampling probe dispenses asample of the final regenerative cell suspension directly into thetube(s) or well(s) within the aggregometer, (5) the tube(s) or well(s)and its contents incubate for an appropriate, designated period of timeunder controlled temperature, (6) the aggregometer reads the contents ofthe tube(s) or well(s), and (7) platelet aggregation activity of thesample is then displayed digitally on the outside of the device.

According to further implementations, In another implementation of theinvention, one or more of the above tests are not automaticallyperformed but rather are automatically displayed (or otherwise conveyedto the user) by the system to remind or prompt the user of the optionfor manual performance thereof.

The tests displayed can be based upon the type of application (e.g.,intravascular delivery vs. non-systemic implantation) input by the user,whereby as described above the system automatically selects (e.g., froma stored set of tests of which the system is capable of accommodating,facilitating or at least partially performing) a group of tests, wherebythe selection can be based upon (i) the type of application and/or (ii)the type of tissue input by the user. The system then automaticallydisplays (or otherwise conveys to the user) these tests and, optionally,prompts the user to choose from among the displayed tests.

A user can provide an input indicating the type of tissue to beprocessed and the application (e.g., the type of tissue to be formed).Based upon that input, the system automatically determines what typestests will be performed. These tests can include measuring forclinically unsafe levels, relative to the application (e.g., type oftissue formation to be induced), of at least one of endotoxins, residualenzymes from for example the digestion, free lipid, and residualextracellular matrix which may be present from for example thedigestion. In a modified embodiment, the system automatically selects(from a stored set of tests of which the system is capable ofperforming) a group of tests, whereby the selection is based upon theinput. The system then automatically displays these tests and promptsthe user to choose from among the displayed tests.

In the case of an intravascular delivery of at least part of thecomposition into the patient, at least part of the composition can be inone example automatically tested for clinically unsafe levels of atleast one of endotoxins, residual enzymes, free lipid, and agonists ofplatelet aggregation. In an exemplary embodiment, the displayed testsinclude these three items, and the user then inputs the particular testswhich are desired to be or should be performed by the automated system.

In the case of a non-systemic implantation of at least part of thecomposition into the patient, agonists of platelet aggregation may notautomatically be tested for clinically unsafe levels. In an exemplaryembodiment, the displayed tests do not include an option for testingparts of the composition for agonists of platelet aggregation.

According to another implementation, in the case of a non-systemicimplantation of at least part of the composition into the patient, freelipid may not be automatically tested for clinically unsafe levels. Inan exemplary embodiment, the displayed tests do not include an optionfor testing parts of the composition for free lipid.

In another preferred embodiment, the system is automated such that theentire method from separation to concentration of the clinically saferegenerative cells may be performed in a continuous sequence withminimal user intervention. Preferably, the entire procedure from tissueextraction through separating, concentrating and placement of theregenerative cells into the recipient would all be performed in the samefacility, indeed, even within the same room of the patient undergoingthe procedure. The regenerative cells may be used in a relatively shorttime period after extraction and concentration. For example, theregenerative cells may be ready for use in about one hour from theharvesting of tissue from a patient, and in certain situations, may beready for use in about 10 to 40 minutes from the harvesting of thetissue. In a preferred embodiment, the regenerative cells may be readyto use in about 20 minutes from the harvesting of tissue. The entirelength of the procedure from extraction through separating andconcentrating may vary depending on a number of factors, includingpatient profile, type of tissue being harvested and the amount ofregenerative cells required for a given therapeutic application. Thecells may also be placed into the recipient in combination with othercells, tissue, tissue fragments, scaffolds or other stimulators of cellgrowth and/or differentiation in the context of a single operativeprocedure with the intention of deriving a therapeutic, structural, orcosmetic benefit to the recipient. It is understood that any furthermanipulation of the regenerative cells beyond the separating andconcentrating phase of the system will require additional timecommensurate with the manner of such manipulation.

Patients suffering from wide variety of diseases and disorders maybenefit from the regenerative cells of the present invention. Forexample, patients suffering from cardiovascular diseases and disorders,liver diseases and disorders, renal diseases and disorders, skeletalmuscle disorders, lung injuries and disorders, diabetes, intestinaldiseases and disorders, nervous system disorders, Parkinson's disease,Alzheimer's, stroke related diseases and disorders, diseases anddisorders of the hematopoietic system, wounds, ulcers and other diseasesand disorders of the skin, traumatic injury, burn, radiation or chemicalor other toxin-induced injuries or disorders, and bone and cartilagerelated diseases and disorders can be treated using the regenerativecells obtained through the systems and methods of the present invention.

In particular embodiments, diseases and disorders that are mediated byangiogenesis and arteriogenesis can be treated with the regenerativecells obtained using the systems and methods of the present invention.For example, acute myocardial infarctions, ischemic cardiomyopathy,peripheral vascular disease, ischemic stroke, acute tubular necrosis,ischemic wounds, sepsis, ischemic bowel disease, diabetic retinopathy,neuropathy, nephropathy, vasculitidies, ischemic encephalopathy,erectile dysfunction, ischemic and/or traumatic spinal cord injuries,multiple organ system failures, ischemic gum disease and transplantrelated ischemia can be treated.

Furthermore, diseases and disorders affecting more than onephysiological system, e.g., traumatic injury involving both soft andhard tissues, the effects of aging, multi-organ disorders, etc., mayalso be treated with the regenerative cells obtained using the systemsand methods of the present invention. The regenerative cells can also beused to promote tendon and cartilage repair and for a variety ofclinical and non-clinical cosmetic and structural applications,including autologous fat transfer applications. Cosmetic applicationsinclude, for example, restructuring of facial folds and wrinkles, lip,breast and buttocks as well as other soft tissue defects. Theregenerative cells may also be used for tissue engineering applicationsknown in the art.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, “regenerative cells” refers to any heterogeneous orhomologous cells obtained using the systems and methods of the presentinvention which cause or contribute to complete or partial regeneration,restoration, or substitution of structure or function of an organ,tissue, or physiologic unit or system to thereby provide a therapeutic,structural or cosmetic benefit. Examples of regenerative cells include:ASCs, endothelial cells, endothelial precursor cells, endothelialprogenitor cells, macrophages, fibroblasts, pericytes, smooth musclecells, preadipocytes, differentiated or de-differentiated adipocytes,keratinocytes, unipotent and multipotent progenitor and precursor cells(and their progeny), and lymphocytes.

One mechanism by which the regenerative cells may provide a therapeutic,structural or cosmetic benefit is by incorporating themselves or theirprogeny into newly generated, existing or repaired tissues or tissuecomponents. For example, ASCs and/or their progeny may incorporate intonewly generated bone, muscle, or other structural or functional tissueand thereby cause or contribute to a therapeutic, structural or cosmeticimprovement. Similarly, endothelial cells or endothelial precursor orprogenitor cells and their progeny may incorporate into existing, newlygenerated, repaired, or expanded blood vessels to thereby cause orcontribute to a therapeutic, structural or cosmetic benefit.

Another mechanism by which the regenerative cells may provide atherapeutic, structural or cosmetic benefit is by expressing and/orsecreting molecules, e.g., growth factors, that promote creation,retention, restoration, and/or regeneration of structure or function ofa given tissue or tissue component. For example, regenerative cells mayexpress and/or secrete molecules which result in enhanced growth oftissues or cells that then participate directly or indirectly inimproved structure or function. Regenerative cells may express and/orsecrete growth factors, including, for example, Vascular EndothelialGrowth Factor (VEGF), Placental Growth factor (PlGF), bFGF, IGF-II,Eotaxin, G-CSF, GM-CSF, IL-12 p40/p70, IL-12 p70, IL-13, IL-6, IL-9,Leptin, MCP-1, M-CSF, MIG, PF-4, TIMP-1, TIMP-2, TNF-α, Thrombopoetin,and their isoforms, which may perform one or more of the followingfunctions: stimulate development of new blood vessels, i.e., promoteangiogenesis; improve oxygen supply of pre-existent small blood vessels(collaterals) by expanding their blood carrying capacity; inducemobilization of regenerative cells from sites distant from the site ofinjury to thereby enhance the homing and migration of such cells to thesite of injury; stimulate the growth and/or promote the survival ofcells within a site of injury thereby promoting retention of function orstructure; deliver molecules with anti-apoptotic properties therebyreducing the rate or likelihood of cell death and permanent loss offunction; and interact with endogenous regenerative cells and/or otherphysiological mechanisms.

The regenerative cells may be used in their ‘native’ form as present inor separated and concentrated from the tissue using the systems andmethods of the present invention or they may be modified by stimulationor priming with growth factors or other biologic response modifiers, bygene transfer (transient or stable transfer), by furthersub-fractionation of the resultant population on the basis or physicalproperties (for example size or density), differential adherence to asolid phase material, expression of cell surface or intracellularmolecules, cell culture or other ex vivo or in vivo manipulation,modification, or fractionation as further described herein. Theregenerative cells may also be used in combination with other cells ordevices such as synthetic or biologic scaffolds, materials or devicesthat deliver factors, drugs, chemicals or other agents that modify orenhance the relevant characteristics of the cells as further describedherein.

As used herein, “regenerative cell composition” refers to thecomposition of cells typically present in a volume of liquid after atissue, e.g., adipose tissue, is washed and at least partiallydisaggregated. For example, a regenerative cell composition of theinvention comprises multiple different types of regenerative cells,including ASCs, endothelial cells, endothelial precursor cells,endothelial progenitor cells, macrophages, fibroblasts, pericytes,smooth muscle cells, preadipocytes, differentiated or de-differentiatedadipocytes, keratinocytes, unipotent and multipotent progenitor andprecursor cells (and their progeny), and lymphocytes. The regenerativecell composition may also contain one or more contaminants, such ascollagen, which may be present in the tissue fragments, or residualcollagenase or other enzyme or agent employed in or resulting from thetissue disaggregation process described herein.

As used herein, “regenerative medicine” refers to any therapeutic,structural or cosmetic benefit that is derived from the placement,either directly or indirectly, of regenerative cells into a subject.Regenerative medicine encompasses all of the diseases and disordersdescribed herein as well as those known in the art.

As used herein, “stem cell” refers to a multipotent regenerative cellwith the potential to differentiate into a variety of other cell types,which perform one or more specific functions and have the ability toself-renew. Some of the stem cells disclosed herein may be multipotent.

As used herein, “progenitor cell” refers to a multipotent regenerativecell with the potential to differentiate into more than one cell typeand has limited or no ability to self-renew. “Progenitor cell”, as usedherein, also refers to a unipotent cell with the potential todifferentiate into only a single cell type, which performs one or morespecific functions and has limited or no ability to self-renew. Inparticular, as used herein, “endothelial progenitor cell” refers to amultipotent or unipotent cell with the potential to differentiate intovascular endothelial cells.

As used herein, “precursor cell” refers to a unipotent regenerative cellwith the potential to differentiate into one cell type. Precursor cellsand their progeny may retain extensive proliferative capacity, e.g.,lymphocytes and endothelial cells, which can proliferate underappropriate conditions.

As used herein “stem cell number” or “stem cell frequency” refers to thenumber of colonies observed in a clonogenic assay in which adiposederived cells (ADC) are plated at low cell density (<10,000 cells/well)and grown in growth medium supporting MSC growth (for example, DMEM/F12medium supplemented with 10% fetal calf serum, 5% horse serum, andantibiotic/antimycotic agents). Cells are grown for two weeks afterwhich cultures are stained with hematoxylin and colonies of more than 50cells are counted as CFU-F. Stem cell frequency is calculated as thenumber of CFU-F observed per 100 nucleated cells plated (for example; 15colonies counted in a plate initiated with 1,000 nucleated regenerativecells gives a stem cell frequency of 1.5%). Stem cell number iscalculated as stem cell frequency multiplied by the total number ofnucleated ADC cells obtained. A high percentage (˜100%) of CFU-F grownfrom regenerative cells express the cell surface molecule CD105 which isalso expressed by marrow-derived stem cells (Barry et al., 1999). CD105is also expressed by adipose tissue-derived stem cells (Zuk et al.,2002).

As used herein, the term “adipose tissue” refers to fat including theconnective tissue that stores fat. Adipose tissue contains multipleregenerative cell types, including ASCs and endothelial progenitor andprecursor cells.

As used herein, the term “unit of adipose tissue” refers to a discreteor measurable amount of adipose tissue. A unit of adipose tissue may bemeasured by determining the weight and/or volume of the unit. Based onthe data identified above, a unit of processed lipoaspirate, as removedfrom a patient, has a cellular component in which at least 0.1% of thecellular component is stem cells; that is, it has a stem cell frequency,determined as described above, of at least 0.1%. In reference to thedisclosure herein, a unit of adipose tissue may refer to the entireamount of adipose tissue removed from a patient, or an amount that isless than the entire amount of adipose tissue removed from a patient.Thus, a unit of adipose tissue may be combined with another unit ofadipose tissue to form a unit of adipose tissue that has a weight orvolume that is the sum of the individual units.

As used herein, the term “portion” refers to an amount of a materialthat is less than a whole. A minor portion refers to an amount that isless than 50%, and a major portion refers to an amount greater than 50%.Thus, a unit of adipose tissue that is less than the entire amount ofadipose tissue removed from a patient is a portion of the removedadipose tissue.

As used herein, the term “processed lipoaspirate” refers to adiposetissue that has been processed to separate the active cellular component(e.g., the component containing regenerative) from the mature adipocytesand connective tissue. This fraction is referred to herein as“adipose-derived cells” or “ADC.” Typically, ADC refers to the pellet ofregenerative cells obtained by washing and separating and concentratingthe cells from the adipose tissue. The pellet is typically obtained bycentrifuging a suspension of cells so that the cells aggregate at thebottom of a centrifuge chamber or cell concentrator.

As used herein, the terms “administering,” “introducing,” “delivering,”“placement” and “transplanting” are used interchangeably herein andrefer to the placement of the regenerative cells of the invention into asubject by a method or route which results in at least partiallocalization of the regenerative cells at a desired site. Theregenerative cells can be administered by any appropriate route whichresults in delivery to a desired location in the subject where at leasta portion of the cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a disease or disorder

As used herein, “therapeutically effective dose of regenerative cells”refers to an amount of regenerative cells that are sufficient to bringabout a beneficial or desired clinical effect. Said dose could beadministered in one or more administrations. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including, but not limited to,the patient's age, size, type or extent of disease, stage of thedisease, route of administration of the regenerative cells, the type orextent of supplemental therapy used, ongoing disease process and type oftreatment desired (e.g., aggressive vs. conventional treatment).

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the subjectis a human.

As previously set forth herein, regenerative cells, e.g., stem andprogenitor cells, can be harvested from a wide variety of tissues. Thesystem of the present invention may be used for all such tissues.Adipose tissue, however, is an especially rich source of regenerativecells. Accordingly, the system of the present invention is illustratedherein using adipose tissue as a source of regenerative cells by way ofexample only and not limitation.

Adipose tissue can be obtained by any method known to a person ofordinary skill in the art. For example, adipose tissue may be removedfrom a patient by liposuction (syringe or power assisted) or bylipectomy, e.g., suction-assisted lipoplasty, ultrasound-assistedlipoplasty, and excisional lipectomy or combinations thereof. Theadipose tissue is removed and collected and may be processed inaccordance with any of the embodiments of a system of the inventiondescribed herein. The amount of tissue collected depends on numerousfactors, including the body mass index and age of the donor, the timeavailable for collection, the availability of accessible adipose tissueharvest sites, concomitant and pre-existing medications and conditions(such as anticoagulant therapy), and the clinical purpose for which thetissue is being collected. For example, the regenerative cell percentageof 100 ml of adipose tissue extracted from a lean individual is greaterthan that extracted from an obese donor (Table 1). This likely reflectsa dilutive effect of the increased fat content in the obese individual.Therefore, it may be desirable, in accordance with one aspect of theinvention, to obtain larger amounts of tissue from overweight donorscompared to the amounts that would be withdrawn from leaner patients.This observation also indicates that the utility of this invention isnot limited to individuals with large amounts of adipose tissue.

TABLE 1 Effect of Body Mass Index on Tissue and Cell Yield Amount ofTissue Total Regenerative Body Mass Index Status Obtained (g) Cell Yield(×10⁷) Normal  641 ± 142 2.1 ± 0.4 Obese 1,225 ± 173 2.4 ± 0.5 p value0.03 0.6

After the adipose tissue is processed, the resulting regenerative cellsare substantially free from free lipid, blood components, matureadipocytes and connective tissue. Accordingly, the system of the presentinvention generates a heterogeneous plurality of adipose derivedregenerative cells which may be used for research and/or therapeuticpurposes. In a preferred embodiment, the cells are clinically safe,i.e., suitable for placement or re-infusion within the body of arecipient. In other embodiments, the cells may be used for research,e.g., the cells can be used to establish stem or progenitor cell lineswhich can survive for extended periods of time and be used for furtherstudy.

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same or similar referencenumbers are used in the drawings and the description to refer to thesame or like parts. It should be noted that the drawings are insimplified form and are not to precise scale. In reference to thedisclosure herein, for purposes of convenience and clarity only,directional terms, such as, top, bottom, left, right, up, down, over,above, below, beneath, rear, front, distal, and proximal are used withrespect to the accompanying drawings. Such directional terms should notbe construed to limit the scope of the invention in any manner.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation. The intent of thefollowing detailed description, although discussing exemplaryembodiments, is to be construed to cover all modifications,alternatives, and equivalents of the embodiments as may fall within thespirit and scope of the invention as defined by the appended claims. Thepresent invention may be utilized in conjunction with various medicalprocedures that are conventionally used in the art.

Referring now to the Figures, a system 10 of the present invention isgenerally comprised of one or more of a tissue collection chamber 20, aprocessing chamber 30, a waste chamber 40, an output chamber 50 and asample chamber 60. The various chambers are coupled together via one ormore conduits 12 such that fluids containing biological material maypass from one chamber to another while maintaining a closed orfunctionally closed, sterile fluid/tissue pathway. A functionally closedpathway refers to a system in which penetration of an otherwisestructurally closed system of bags, tubing, and other components is madesolely in an aseptic or sterile fashion. Typically, this includesadditional of materials through a sealed rubber septum that has beencleaned by wiping with alcohol, povidone iodine or similar agent,through a luer lock-type fitting in an aseptic or sterile environment,or through a temporary opening that, while open, is maintained within anaseptic or sterile environment. Use of sterile connecting devices inwhich one closed system is attached to a second closed system in aclosed, sterile or aseptic fashion is also typical of a functionallyclosed system.

The conduits may comprise rigid or flexible bodies referred tointerchangeably herein as lumens and tubing, respectively. In certainembodiments, the conduits are in the form of flexible tubing, such aspolyethylene tubing conventionally used in clinical settings, siliconeor any other material known in the art. The conduits 12 can vary in sizedepending on whether passage of fluid or tissue is desired. The conduits12 may also vary in size depending on the amount of tissue or fluid thatis cycled through the system. For example, for the passage of fluid, theconduits may have a diameter ranging from about 0.060 to about 0.750inches and for the passage of tissue, the conduits may have a diameterranging from 0.312 to 0.750 inches. Generally, the size of the conduitsis selected to balance the volume the conduits can accommodate and thetime required to transport the tissue or fluids through said conduits.In automated embodiments of the system, the foregoing parameters, i.e.,volume and time for transport, must be identified such that theappropriate signals can be transmitted to the processing device of thesystem. This allows the device to move accurate volumes of liquid andtissue from one chamber to another. The flexile tubing used should becapable of withstanding negative pressure to reduce the likelihood ofcollapse. The flexible tubing used should also be capable ofwithstanding positive pressure which is generated by, for example, apositive displacement pump, which may be used in the system.

All the chambers of the system may be comprised of one or more ports,e.g., outlet 22 or inlet 21 ports, which accept standard IV, syringe andsuction tubing connections. The ports may be a sealed port such as arubber septum closed syringe needle access port 51. The inlet ports maybe coupled to one or more cannulas (not shown) by way of conduits. Forexample, a tissue inlet port 21 may be coupled to an integrated singleuse liposuction cannula and the conduit may be a flexible tubing. Theconduits are generally positioned to provide fluid passageways from onechamber of the system to another. Towards this end, the conduits andports may be coupled to, for example, a suction device (not shown) whichmay be manually or automatically operated. The suction device may be,e.g., a syringe or an electric pump. The suction device should becapable of providing sufficient negative pressure to aspirate tissuefrom a patient. Generally, any suitable suction device known to one ofordinary skill in the art, e.g., a surgeon, may be used.

The conduits 12 may further comprise one or more clamps (not shown) tocontrol the flow of material among various components of the system. Theclamps are useful for maintaining the sterility of the system byeffectively sealing different regions of the system. Alternatively, theconduits 12 may comprise one or more valves 14 that control the flow ofmaterial through the system. The valves 14 are identified as opencircles in the Figures. In preferred embodiments, the valves may beelectromechanical pinch valves. In another embodiment, the valves may bepneumatic valves. In yet other embodiments, the valves may be hydraulicvalves or mechanical valves. Such valves are preferably activated by acontrol system which may be coupled to levers. The levers may bemanually manipulated such that the levers are activated. In automatedembodiments, the control system may be coupled to the levers as well asto a processing device which may activate the valves at pre-determinedactivation conditions. In certain automated embodiments, activation ofthe valves may be partially automated and partially subject to theuser's preference such that the process may be optimized. In yet otherembodiments, certain valves may be activated manually and othersautomatically through the processing device. The valves 14 may also beused in conjunction with one or more pumps, e.g., peristaltic pumps 34or positive displacement pumps (not shown). The conduits 12 and/or thevalves 14 may also be comprised of sensors 29, e.g., optical sensors,ultrasonic sensors, pressure sensors or other forms of monitors known inthe art that are capable of distinguishing among the various fluidcomponents and fluid levels that flow through the system. In a preferredembodiment, the sensors 29 may be optical sensors.

The system may also include a plurality of filters 36. In certainembodiments, the filters may be within a chamber of the system 28.Different chambers within the system may be comprised of differentfilters. The filters are effective to separate the regenerative cells,e.g., stem cells and/or progenitor cells, from undesirable cells anddisaggregation agents that may be used in accordance with the system. Inone embodiment, a filter assembly 36 includes a hollow fiber filtrationdevice. In another embodiment, a filter assembly 36 includes apercolative filtration device, which may or may not be used with asedimentation process. In a further embodiment, the filter assembly 36comprises a centrifugation device, which may or may not be used with anelutriation device and process. In yet another embodiment, the systemcomprises a combination of these filtering devices. The filtrationfunctions of the present invention can be two-fold, with some filtersremoving things from the final concentration such as collagen, freelipid, free adipocytes and residual collagenase, and with other filtersbeing used to concentrate the final product. The filters of the systemmay be comprised of a plurality of pores ranging in diameters and/orlength from 20 to 800 μm. In a preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of pores rangingfrom 80 to 400 μm. In another preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of 265 μm pores. Inother embodiments, the filters may be detachable and/or disposable.

The system may also be comprised of one or more temperature controldevices (not shown) that are positioned to adjust the temperature of thematerial contained within one or more chambers of the system. Thetemperature control device may be a heater, a cooler or both, i.e., itmay be able to switch between a heater and a cooler. The temperaturedevice may adjust the temperature of any of the material passing throughthe system, including the tissue, the disaggregation agents, theresuspension agents, the rinsing agents, the washing agents or theadditives. For example, heating of adipose tissue facilitatesdisaggregation whereas the cooling of the regenerative cell output isdesirable to maintain viability. Also, if pre-warmed reagents are neededfor optimal tissue processing, the role of the temperature device wouldbe to maintain the pre-determined temperature rather than to increase ordecrease the temperature.

To maintain a closed, sterile fluid/tissue pathway, all ports and valvesmay comprise a closure that maintains the sealed configuration of thesystem. The closure may be a membrane that is impermeable to fluid, airand other contaminants or it may be any other suitable closure known inthe art. Furthermore, all ports of the system may be designed such thatthey can accommodate syringes, needles or other devices for withdrawingthe materials in the chambers without compromising the sterility of thesystem.

As set forth herein, tissue may be extracted from a patient via any artrecognized method. The aspirated tissue may be extracted prior to beingplaced in the system for processing. The aspirated tissue is typicallytransferred to the collection chamber 20 through conduits 12 via asealed entry port, such as a rubber septum closed syringe needle accessport (not shown on collection chamber). Alternatively, the tissueextraction step may be part of the system. For example, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula inserted into the patient. Thus, inthis embodiment, the entire system is attached to the patient. Thetissue may be introduced into the collection chamber 20 through an inletport 21 via a conduit such as 12 a which are part of a closed sterilepathway. The collection chamber 20 may be comprised of a plurality offlexible or rigid canisters or cylinders or combinations thereof. Forexample, the collection chamber 20 may be comprised of one or more rigidcanisters of varying sizes. The collection chamber 20 may also becomprised of one or more flexible bags. In such systems, the bag ispreferably provided with a support, such as in internal or externalframe, that helps reduce the likelihood that the bag will collapse uponthe application of suction to the bag. The collection chamber 20 issized to hold the requisite amount of saline to appropriately wash anddisaggregate the tissue prior to the wash and concentrate stage of theprocess performed in the processing chamber 30. Preferably, the volumeof tissue or fluid present in the collection chamber 20 is easilyascertainable to the naked eye. For example, to obtain regenerativecells from adipose tissue, a suitable collection chamber has thecapacity to hold 800 ml of lipoaspirate and 1200 ml of saline.Accordingly, in one embodiment, the collection chamber 20 has a capacityof at least 2 liters. In another embodiment, to separate and concentratered blood cells from blood, the collection chamber 20 has a capacity ofat least 1.5 liters. Generally, the size of the collection chamber 20will vary depending on the type and amount of tissue collected from thepatient. The collection chamber 20 may be sized to hold as little asabout 5 ml to up to about 2 liters of tissue. For smaller tissuevolumes, e.g., 5 mls to 100 mls, the tissue may be gathered in a syringeprior to transfer to the collection chamber 20.

The collection chamber 20 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the collection chamber 20 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact as described in the ISO 10993 standard. For example,polycarbonate acrylic or ABS may be used. The fluid path of thecollection chamber 20 is preferably pyrogen free, i.e., suitable forblood use without danger of disease transmittal. In one embodiment, thecollection chamber 20 is constructed of a material that allows the userto visually determine the approximate volume of tissue present in thechamber. In other embodiments, the volume of tissue and/or fluid in thecollection chamber 20 is determined by automated sensors 29. Thecollection chamber 20 is preferably designed such that in an automatedembodiment, the system can determine the volume of tissue and/or fluidwithin the chamber with a reasonable degree of accuracy. In a preferredembodiment, the system senses the volume within the collection chamberwith an accuracy of plus or minus fifteen percent.

In a particular embodiment provided by way of example only, thecollection chamber 20 is in the form of a rigid chamber, for example, achamber constructed of a medical grade polycarbonate containing aroughly conical prefixed filter 28 of medical grade polyester with amesh size of 265 μm (see FIG. 5). The rigid tissue collection containermay have a size of approximately eight inches high and approximatelyfive inches in diameter; the wall thickness may be about 0.125 inches.The interior of the cylinder may be accessed through, for example, oneor more ports for suction tubing, one or more ports with tubing forconnection through sterile docking technology, and/or one or more portsfor needle puncture access through a rubber septum. The prefixed filter28 in the interior of the collection chamber 20 is preferably structuredto retain adipose tissue and to pass non-adipose tissue as, for example,the tissues are removed from the patient. More specifically, the filter28 may allow passage of free lipid, blood, and saline, while retainingfragments of adipose tissue during, or in another embodiment after, theinitial harvesting of the adipose tissue. In that regard, the filter 28includes a plurality of pores, of either the same or different sizes,but ranging in size from about 20 μm to 5 mm. In a preferred embodiment,the filter 28 includes a plurality of 400 μm pores. In a preferredembodiment, the filter 28 is a medical grade polyester mesh of around200 μm thickness with a pore size of around 265 μm and around 47% openarea. This material holds the tissue during rinsing but allows cells topass out through the mesh following tissue disaggregation. Thus, whenthe tissues are aspirated from the patient, non-adipose tissue may beseparated from adipose tissue. The same functionality could be achievedwith different materials, mesh size, and the number and type of ports.For example, mesh pore sizes smaller than 100 μm or as large as severalthousand microns would achieve the same purpose of allowing passage ofsaline and blood cells while retaining adipose tissue aggregates andfragments. Similarly, the same purpose could be achieved by use of analternative rigid plastic material, or by many other modifications thatwould be known to those skilled in the art

The system 10 may also be comprised of one or more solution sources 22.The solution source may comprise a washing solution source 23, and atissue disaggregation agent source 24, such as collagenase. Thecollection chamber 20 is comprised of closed fluid pathways that allowsfor the washing and disaggregating solutions or agents to be added tothe tissue in an aseptic manner.

The containers for the washing solution 23 and the disaggregation agents24 may be any suitable container that can hold their contents in asterile manner, e.g., a collapsible bag, such as an IV bag used inclinical settings. These containers may have conduits 12, such asconduit 12 e, coupled to the collection chamber 20 so that the washingsolution and the disaggregation agent may be delivered to the interiorof the collection chamber 20. The washing solution and thedisaggregation agent may be delivered to the interior of the collectionchamber 20 through any art-recognized manner, including simple gravitypressure applied to the outside of the containers for the saline 23and/or the disaggregation agents 24 or by placement of a positivedisplacement pump on the conduits, e.g., conduit 12 d in FIG. 4. Inautomated embodiments, the processing device of the system calculatesvarious parameters, e.g., the volume of saline and time or number ofcycles required for washing as well as the concentration or amount ofdisaggregation agent and the time required for disaggregation based oninformation initially entered by the user (e.g., volume of tissue beingprocessed). Alternatively, the amounts, times etc. can be manuallymanipulated by the user.

The tissue and/or fluid within the collection chamber should bemaintained at a temperature ranging from 30 degrees Celsius to 40degrees Celsius. In a preferred embodiment, the temperature of thesuspension inside the collection chamber is maintained at 37 degreesCelsius. In certain embodiments, if the surgical procedure ortherapeutic application needs to be delayed, the selected tissue may bestored in the collection chamber for later use. The tissue may be storedat or about room temperature or at about 4 degrees Celsius for up to 96hours.

The washing solution may be any solution known to one of skill in theart, including saline or any other buffered or unbuffered electrolytesolution. The types of tissue being processed will dictate the types orcombinations of washing solutions used. Typically, the washing solution,such as saline, enters the collection chamber 20 after the adiposetissue has been removed from the patient and placed in the collectionchamber. However, the washing solution may be delivered to thecollection chamber 20 before the adipose tissue is extracted, or may bedelivered to the collection chamber 20 concurrently with the adiposetissue. In the collection chamber 20, the washing solution and theextracted adipose tissue may be mixed by any means including the methodsdescribed below.

For example, the tissue may be washed by agitation (which maximizes cellviability and minimizes the amount of free lipid released). In oneembodiment, the tissue is agitated by rotating the entire collectionchamber 20 through an arc of varying degrees (e.g., through an arc ofabout 45 degrees to about 90 degrees) at varying speeds, e.g., about 30revolutions per minute. In other embodiments, the tissue is agitated byrotating the entire collection chamber 20, wherein the collectionchamber 20 is comprised of one or more paddles or protrusions rigidlyattached to an inside surface of the collection chamber, through an arcof varying degrees (e.g., through an arc of about 45 degrees to about 90degrees) at varying speeds, e.g., about 30 revolutions per minute. Therotation of the collection chamber 20 described above may beaccomplished by a drive mechanism attached to or in proximity with thecollection chamber 20. The drive mechanism may be a simple belt or gearor other drive mechanism known in the art. The speed of the rotation maybe, for example, 30 revolutions per minute. Generally, higher speedshave been found to generate larger volumes of free lipids and may not beoptimal.

In other embodiments, the tissue is agitated by placing a rotatableshaft 25 inside the collection chamber 20, wherein the rotatable shaftis comprised of one or more paddles 25 a or protrusions rigidly attachedto the rotatable shaft 25 which pass through the mixture as the shaft isbeing rotated. In certain embodiments, the rotatable shaft 25 withrigidly attached 25 a paddles may be rested on the bottom of thecollection chamber 20. This may be accomplished, for example, by placingthe paddle-like device into a spinning magnetic field (e.g., magneticstirrer). Alternatively, agitating of the tissue may be accomplishedusing a simple agitator known in the art, i.e. a device implementingshaking up and down without rotation. The tissue may also be washedusing any other art-recognized means including rocking, stirring,inversion, etc.

After a desired amount of wash cycles, a tissue disaggregation agent maybe delivered to the collection chamber 20 to separate the regenerativecells from the remaining adipose tissue components. The disaggregationagent may be any disaggregation agent known to one of skill in the art.Disaggregation agents that may be used include neutral proteases,collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, membersof the Blendzyme enzyme mixture family, e.g., Liberase H1, pepsin,ultrasonic or other physical energy, lasers, microwaves, othermechanical devices and/or combinations thereof. A preferreddisaggregation agent of the invention is collagenase. In a preferredembodiment, the disaggregation agents used will be approved for humanuse by the relevant authority (e.g., the U.S. Food and DrugAdministration). In all embodiments, the disaggregation agents will befree of viable micro-organisms and other contaminants, such asendotoxin.

The disaggregation agents may be added with other solutions. Forexample, saline, such as saline delivered from a saline source 23 asdescribed above, may be added to the adipose tissue along with orimmediately followed by addition of collagenase. In one embodiment, thewashed adipose tissue is mixed with a collagenase-containing enzymesolution at or around 37° C. for about 20-60 minutes. In one particularembodiment, the tissue is washed with sterile buffered isotonic salineand incubated with a combination of disaggregation agents such ascollagenases and neutral proteases at a concentration, temperature, andtime sufficient to provide adequate disaggregation. Suitable neutralproteases are obtainable may be obtained from F. Hoffmann-La Roche Ltd,Indianapolis, Ind. Suitable collagenase preparations include recombinantand non-recombinant collagenase. Non-recombinant collagenase may beobtained from F. Hoffmann-La Roche Ltd, Indianapolis, Ind. and/orAdvance Biofactures Corp., Lynbrook, N.Y. Recombinant collagenase mayalso be obtained as disclosed in U.S. Pat. No. 6,475,764.

In a preferred embodiment, the washed adipose tissue is combined with anenzyme cocktail, Blendzyme 3® (Roche Diagnostics), to yield thefollowing concentration of enzymes: Collagenase I and II (0.5 Wunschunits/ml) and Thermolysin (241 Caseinase units/ml). The tissue is theincubated at 37° C. for 15-25 minutes. These parameters will varyaccording to the source of the collagenase enzyme, optimized byempirical studies, in order to validate that the system is effective atextracting the desired cell populations in an appropriate time frame. Ina particularly preferred embodiment the enzyme(s) used is materialapproved for human use by the relevant authority (e.g., the U.S. Foodand Drug Administration).

In other embodiments, a higher concentration of collagenase or similaragent may be added to decrease the digestion time. The washed adiposetissue and the tissue disaggregation agent may then be agitated inmanners similar to the agitation methods described above, until thewashed adipose tissue is disaggregated. For example, the washed adiposetissue and the tissue disaggregation agent may be agitated by rotatingthe entire collection chamber through an arc of approximately 90degrees, by having a shaft which contains one or more paddles which passthrough the solution as the shaft is being rotated, and/or by rotatingthe entire collection chamber which contains paddles or protrusions onthe inside surface of the collection chamber.

Depending on the purpose for which the adipose derived cells will beused, the adipose tissue may either be partially disaggregated, orcompletely disaggregated. For example, in embodiments in which theadipose derived cells are to be combined with a unit of adipose tissue,it may be desirable to partially disaggregate the harvested adiposetissue, to remove a portion of the partially disaggregated adiposetissue, and then continue disaggregating the remaining portion ofadipose tissue remaining in the collection chamber. Alternatively, aportion of washed adipose tissue may be removed and set aside in asample container prior to any digestion. In another embodiment,harvested adipose tissue is partially disaggregated to concentrate cellsbefore being reintroduced back into the patient. In one embodiment, theadipose tissue is mixed with a tissue disaggregation agent for a periodof time generally less than about 20 minutes. A portion of the partiallydisaggregated tissue may then be removed from the collection chamber,and the remaining partially disaggregated tissue may be furtherdisaggregated by mixing the adipose tissue with a tissue disaggregationagent for another 40 minutes. When the adipose derived cells are to beused as an essentially pure population of regenerative cells, theadipose tissue may be fully disaggregated.

After digestion, the tissue and disaggregation agent solution is allowedto settle for a period of time sufficient to allow the buoyant andnon-buoyant components of the solution to differentiate within thecollection chamber. Typically, the time ranges from about 15 seconds toseveral minutes but other times may be implemented in modifiedembodiments. The buoyant layer is comprised of the regenerative cellsthat require further washing and concentrating. The non-buoyant layercomprises blood, collagen, lipids and other non-regenerative cellcomponents of the tissue. The non-buoyant layer must be removed to thewaste chamber.

Accordingly, the collection chamber 20 is preferably comprised of anoutlet port 22 at the lowest point of the chamber such that blood andother non-buoyant components of the tissue may be drained to one or morewaste containers 40 via one or more conduits 12. The collection chamber20 is generally in (or may be placed in) an upright position such thatthe outlet ports 22 are located at the bottom of the collection chamber.The draining may be passive or active. For example, the non-buoyantcomponents described above could be drained using gravity, by applyingpositive or negative pressure, by use of pumps 34 or by use of vents 32.In automated embodiments, the processing device can signal certainvalves and/or pumps to drain the non-buoyant layer from the collectionchamber 20. The automated embodiments may also be comprised of sensors29 which can detect when the interface between the buoyant andnon-buoyant liquids has been reached. The automated embodiments may alsobe comprised of a sensor 29, e.g., an optical sensor, which may becapable of detecting a change in the light refraction of the effluentwhich is flowing in the conduit leading out of the collection chamber.The appropriate change in the light refraction may signal the presenceof the buoyant layer in the outgoing conduits which indicates that thenon-buoyant layer has been drained. The sensor 29 can then signal theprocessing device to proceed with the next step.

In certain embodiments however, the tissue may be processed to retrievethe non-regenerative cell component of the tissue. For example, incertain therapeutic or research applications, collagen, proteins, matrixor stromal components, lipids, adipocytes or other components of thetissue may be desired. In such embodiments, it is the buoyant layercomprising the regenerative cells that must be removed as describedabove to the waste chamber. The non-buoyant layer is then retained inthe system for further processing as needed.

Once the non-buoyant layer is removed, the buoyant layer comprising theregenerative cells may be washed one or more times to remove residualcontaminants. Accordingly, the collection chamber 20 typically includesone or more ports 21 for permitting the washing solution to be deliveredto the interior of the chamber, and one or more ports 22 for permittingwaste and other materials to be directed out from the collection chamber20. For example, the collection chamber may include one or more sealedentry ports as described herein. The collection chamber 20 may alsoinclude one or more caps (not shown), such as a top cap and a bottom capto further ensure that the system remains sterile while washing solutionis delivered into the collection chamber and/or waste is transportedout. The ports 21 may be provided on the caps of the collection chamberor on a sidewall of the collection chamber.

The process of washing with fresh wash solution may be repeated untilthe residual content of non-buoyant contaminants in the solution reachesa pre-determined level. In other words, the remaining material in thecollection chamber 20, which comprises the buoyant material of themixture described above, including adipose tissue fragments, may bewashed one or more additional times until the amount of undesiredmaterial is reduced to a desired pre-determined level. One method ofdetermining the end point of the washing is to measure the amount of redblood cells in the tissue solution. This can be accomplished bymeasuring the light absorbed on the 540 nm wavelength. In a preferredembodiment, a range between about 0.546 and about 0.842 is deemedacceptable.

During the washing and/or disaggregation, one or more additives may beadded to the various containers as needed to enhance the results. Someexamples of additives include agents that optimize washing anddisaggregation, additives that enhance the viability of the active cellpopulation during processing, anti-microbial agents (e.g., antibiotics),additives that lyse adipocytes and/or red blood cells, or additives thatenrich for cell populations of interest (by differential adherence tosolid phase moieties or to otherwise promote the substantial reductionor enrichment of cell populations). Other possible additives includethose that promote recovery and viability of regenerative cells (forexample, caspase inhibitors) or which reduce the likelihood of adversereaction on infusion or emplacement (for example, inhibitors ofre-aggregation of cells or connective tissue).

After a sufficient settling time has elapsed, the non-buoyant fractionof the resulting mixture of washed adipose tissue fragments and tissuedisaggregation agents will contain regenerative cells, e.g., stem cellsand other adipose derived progenitor cells. As discussed herein, thenon-buoyant fraction containing the regenerative cells will betransferred to the processing chamber 30 wherein the regenerative cellsof interest, such as the adipose derived stem cells, will be separatedfrom other cells and materials present in the non-buoyant fraction ofthe mixture. This non-buoyant fraction is referred to herein as theregenerative cell composition and comprises multiple different types ofcells, including stem cells, progenitor cells, endothelial precursorcells, adipocytes and other regenerative cells described herein. Theregenerative cell composition may also contain one or more contaminants,such as collagen and other connective tissue proteins and fragmentsthereof, which were present in the adipose tissue fragments, or residualcollagenase from the tissue disaggregation process.

The processing chamber 30 of the invention is preferably positionedwithin the system such that the regenerative cell composition moves fromthe collection chamber 20 to the processing chamber 30 by way of tubing12, valves 14 and pump 34 in a sterile manner. The processing chamber issized to accommodate tissue/fluid mixtures ranging from 10 mL to 1.2 L.In a preferred embodiment, the processing chamber is sized toaccommodate 800 mLs. In certain embodiments, the entire regenerativecell composition from the collection chamber 20 is directed to theprocessing chamber 30. However, in other embodiments, a portion of theregenerative cell composition is directed to the processing chamber 30,and another portion is directed to a different region of the system,e.g., the sample chamber 60, to be recombined with cells processed inthe processing chamber 30 at a later time.

The processing chamber 30 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the processing chamber 30 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact, as described in the ISO 10993 standard. For example,polycarbonate, acrylic, ABS, ethylene vinyl acetate or styrene-butadienecopolymers (SBC) may be used. In another embodiment, the fluid path ofthe disposable processing chamber is pyrogen free. The processingchamber may be in the form of a plastic bag, such as thoseconventionally used in processing blood in blood banks; or in otherembodiments, it may be structurally rigid (FIG. 6). In one embodiment,the processing chamber 30 may be similar to the processing chamberdisclosed in commonly owned U.S. application Ser. No. 10/316,127, filedDec. 7, 2001 and U.S. application Ser. No. 10/325,728, filed Dec. 20,2002, the contents of which in their entirety are hereby incorporated byreference.

The processing chamber 30 may be constructed in any manner suitable forseparating and concentrating cells, including filtration andcentrifugation and/or combinations thereof. In certain embodiments, theregenerative cell composition from the collection chamber 20 isintroduced into the processing chamber 30 where the composition can befiltered to separate and/or concentrate a particular regenerative cellpopulation. Cell filtration is a method of separating particularcomponents and cells from other different components or types of cells.For example, the regenerative cell composition of the inventioncomprises multiple different types of cells, including stem cells,progenitor cells and adipocytes, as well as one or more contaminants,such as collagen, which was present in the adipose tissue fragments, orresidual collagenase from the tissue disaggregation process. The filters36 present in the processing chamber 30 may allow for separation andconcentration of a particular subpopulation of regenerative cells, e.g.,stem cells or endothelial progenitors cells etc.

Some variables which are associated with filtration of cells from aliquid include, but are not limited to, pore size of the filter media,geometry (shape) of the pore, surface area of the filter, flow directionof the solution being filtered, trans-membrane pressure, dilution of theparticular cell population, particulate size and shape as well as cellsize and cell viability. In accordance with the disclosure herein, theparticular cells that are desired to be separated or filtered aretypically adipose derived stem cells. However, in certain embodiments,the particular cells may include adipose derived progenitor cells, suchas endothelial precursor cells, alone or in combination with the stemcells.

The regenerative cell composition may be directed through a filterassembly, such as filter assembly 36. In certain embodiments, the filterassembly 36 comprises a plurality of filters which are structured toperform different functions and separate the regenerative cellcomposition into distinct parts or components. For example, one of thefilters may be configured to separate collagen from the regenerativecell composition, one of the filters may be configured to separateadipocytes and/or lipid components from the regenerative cellcomposition, and one of the filters may be configured to separateresidual enzymes, such as the tissue disaggregation agent, from theregenerative cell composition. In certain embodiments, one of thefilters is capable of performing two functions, such as separatingcollagen and the tissue disaggregation agent from the composition. Theplurality of filters are typically serially arranged; however, at leasta portion of the filters may be arranged in parallel, as well. A serialarrangement of the filters of the filter assembly 36 is shown in FIG. 2.A parallel arrangement of the filters of the filter assembly 36 is shownin FIG. 3.

In one embodiment, the filter assembly 36 comprises a first filter, asecond filter, and a third filter. The first filter is configured toremove collagen particles present in the regenerative cell composition.These collagen particles are typically approximately 0.1 microns indiameter and can be up to 20 microns long. The collagen particles may beof varying sizes depending on the digestion. They also may be fibrils,meaning they have twists and turns. Any of the filters described hereinmay be made from polyethersulfone, polyester, PTFE, polypropylene, PVDF,or possibly cellulose. There are two possibilities for filtering thecollagen. One is to try to remove the larger particles first, lettingthe cells go through, which would require for example a filter probablyin the 10 micron range. The second method is to use a smaller sizefilter, such as 4.5 micron, with the intent that the collagen would bewell digested, so as to trap the cells, and let the collagen passthrough. This would require a means to float the cells back off thefilter. There may also be a possibility of implementing a filter whichwould attract and hold the collagen fibers.

The second filter is configured to remove free immature adipocytes whichare not buoyant in the regenerative cell composition. In one embodimentthe second filter can be constructed of polyester and have a pore sizebetween about 30 and about 50 microns with a preferred pore size beingabout 40 microns. Although referred to as a second filter, placement ofsuch a device may be in a first, rather than second, position tofacilitate an initial removal of larger cells and particles. The thirdfilter is configured to remove the unused or residual collagenase orother tissue disaggregation agent present in the composition. In apreferred implementation, the collagenase may degenerate over time. Inone embodiment, the third filter comprises a plurality of pores having adiameter, or length less than 1 μm. In certain embodiments, the poresmay have diameters that are smaller than 1 μm. In other embodiments, thepores have diameters between 10 kD and 5 microns. In certainembodiments, the third filter may be configured to concentrate theregenerative cell population into a small volume of saline or otherwashing solution, as discussed herein. As presently preferred, only thefinal filter is the hollow fiber unit. It is not necessary for any ofthe filters to be of the hollow fiber type. The hollow fiber unit isused for the final filter in a preferred implementation because it isthe most efficient in removing the collagenase with the smallestdetrimental effect to the regenerative cells. In an embodiment whereinthe device is a collection of off the shelf items, the three filters arein separate housings. It is feasible to have the first and secondfilters combined into one housing if a hollow fiber unit is used for thethird filter. If the final filter is not a hollow fiber set-up then allthree filters can be contained in one housing.

The filters of the filter assembly 36 may be located in the processingchamber 30 or may be provided as components separate from the processingchamber 30. In addition, the filters of the filter assembly 36 may beprovided in multiple processing chambers or in an inline fashion. Incertain embodiments, the conduits or tubing may act as a processingchamber or chambers. The processing chamber can be reduced in size suchthat it becomes the inside volume of the conduits which connect thefilters. This type of system will function correctly if the volume oftissue solution is sized appropriately. Thus, the conduits may act asthe processing chamber by containing the fluid with cells as it is beingrun through the filters. Care may be taken to minimize the volume of theconduits so that cells/tissue are not unnecessarily lost in the processof priming and running the system.

Referring to the embodiment described above, the regenerative cellcomposition, containing the washed cells and residual collagen,adipocytes, and/or undigested tissue disaggregation agent, may bedirected through the first filter to remove at least a portion of andpreferably substantially all of the collagen particles from thecomposition so that fewer, and preferably no, collagen particles arepresent in the filtered solution. The filtered regenerative cellcomposition containing the adipocytes and/or undigested tissuedisaggregation agent, may then be directed through the second filter toremove at least a portion of and preferably substantially all of thefree adipocytes from the filtered regenerative cell composition.Subsequently, the twice filtered regenerative cell composition,containing the undigested tissue disaggregation agent, may be directedthrough the third filter, such as a hollow fiber filtration device, asdiscussed herein, to remove or reduce the undigested tissuedisaggregation agent from the regenerative cell composition.

The thrice-filtered regenerative cell composition (i.e., the compositionremaining after being passed through the first, second, and thirdfilters) may then be directed to multiple outlets, which may include aportion of the processing chamber 30 comprising multiple outlets. Theseoutlets can serve to maintain the necessary pressure, as well as toprovide connections via conduits to other containers which may includethe collection chamber 20, the output chamber 50, and/or the wastecontainer 40.

In one embodiment, a filter of the filter assembly 36 comprises ahollow-fiber filtration member. Or, in other words, the filter comprisesa collection of hollow tubes formed with the filter media. Examples offilter media which can be used with the disclosed system 10 includepolysulfone, polyethersulfone or a mixed ester material, and the like.These hollow fibers or hollow tubes of filter media may be contained ina cylindrical cartridge of the filter assembly 36. The individual tubesor fibers of filter media typically have an inside diameter which rangesfrom about 0.1 mm to about 1 mm with a preferred value being about 0.5mm. The diameter and length of a suitable cylindrical cartridge willdetermine the number of individual tubes of filter media which can beplaced inside the cartridge. One example of a suitable hollow fiberfilter cartridge is the FiberFlo® Tangential Flow Filter, catalog#M-C-050-K (Minntech, Minneapolis, Minn.). Pore sizes of the filtermedia can range between about 10 kiloDaltons and about 5 microns with apreferred pore size being about 0.5 microns.

In the hollow-fiber filter, each hollow tube has a body with a firstend, a second end, and a lumen located in the body and extending betweenthe first end and second end. The body of each hollow tube includes aplurality of pores. The pores are generally oriented in the body so thata regenerative cell composition is filtered by flowing through the lumenof the body, and the products to be filtered tangentially pass throughthe pores, as shown in FIG. 12A. In other words, the smaller particlesin the liquid pass tangentially through the pores relative the flow offluid through the lumen of the body. The composition with theregenerative cells passes through the lumen of each hollow tube when thecomposition is being filtered. Preferably, the flow of the compositionis tangential to the pores of the body of each hollow tube.

By using a tangential flow of fluid, the efficiency of filtration of thestem cells may be enhanced relative to other filtration techniques. Forexample, in accordance with some filtration techniques, the pores of thefilter media are placed in such a manner that the filter is orientatedperpendicular to the flow of the fluid so that the Filter media blocksthe path of the fluid being filtered, as illustrated in FIG. 12B. Inthis type of filtration, the particles which are being filtered out ofthe regenerative cell composition, e.g., the stem cells, tend to buildup on one side of the filter and block the flow of the fluid through thepores. This blockage can reduce the efficiency of the filter. Inaddition, the cells are constantly compressed by the pressure of thefluid flow as well as the weight of the cells accumulating on theupstream side of the filter. This can lead to increased lysis of stemcells. Thus, in such filtration techniques wherein the flow of fluid isparallel to the orientation of the pores in the filter, both large cellsand small particles can be undesirably directed against the filter mediaas the fluid is passed through the pores. Consequently, larger productsin the liquid such as cells may block the pores, thereby decreasing thefiltering effect and increasing an occurrence of cell rupture or injury.

In contrast, in the hollow fiber configuration of the present system 10,the fluid which is being filtered flows inside the lumen of the hollowtube. The portion of the fluid which has the ability to pass through thepores of the body of the filter does so with the aid of the positivepressure of the fluid on the inside of the body as well as a negativepressure which is applied on the outside of the body. In thisembodiment, the cells typically are not subjected to the pressure of thefluid flow or the weight of other cells, and therefore, the shear forceson the stem cells are reduced Thus, the efficiency and effectiveness ofthe filtration can be enhanced by the reduction in clogging rates andthe reduction in regenerative cell lysis. Due to the size of the salineand unwanted protein molecules, during filtration, these molecules andother small components pass through the pores of the bodies of thehollow tubes to the outside of the hollow tubes and are directed to thewaste container 40. In one embodiment, filtration is enhanced bygenerating a vacuum on the outside of the hollow tube filter media. Dueto the size of the regenerative cells, e.g., stem cells or progenitorcells, these cells typically cannot pass through the pores of the bodyand therefore remain on the inside of the hollow tube filter (e.g., inthe lumens of the tubes) and are directed back to the processing chamber30 via a conduit between the filter and the processing chamber, or tothe output chamber 50.

In one specific embodiment, the hollow fiber filter has about a 0.05micron pore size, and contains approximately 550 cm² surface area offilter media. An individual media tube typically has a diameter of about0.5 mm. In processing 130 ml of the regenerative cell composition,approximately 120 ml of additional saline may be added to thecomposition. The processing or filter time may be approximately 8minutes. The differential of the pressures on either side of the body ofthe hollow fiber tube (e.g., the pressure inside the lumen of the body,and outside the body) is considered the trans-membrane pressure. Thetrans-membrane pressure can range from about 1 mmHg to about 500 mmHgwith a preferred pressure being about 200 mmHg. The average nucleatedcell recovery and viability using hollow fiber filtration can beapproximately 80% of viable cells.

The amount of collagenase which is typically removed in such a systemequates to a three log reduction. For example if the initialconcentration of collagenase in the regenerative cell composition whichis transferred from the collection chamber to the processing chamber is0.078 U/ml the collagenase concentration of the final regenerative cellcomposition would be 0.00078 U/ml. The collagenase is removed in thehollow fiber filter, and the hollow fiber filter corresponds to thethird filter discussed above.

Processing chambers illustrating one or more cell filtration methodsdescribed above are shown in the Figures, particularly FIGS. 1-3. Withreference to FIGS. 1-3, between the processing chamber 30 and thefiltering chamber of the filter assembly 36, a pump may be provided,such as pump 34. In addition, vent and pressure sensors, such as vent32, and pressure sensor 39, may be provided in line with the processingchamber 30 and the filter assembly 36. Fittings for the output chamber50 may also be provided. These optional components (e.g., the pump 34,the vent 32, the pressure sensor 39, and the fittings for the outputchamber 50) may be provided between the processing chamber 30 and thefilter assembly 36 so that liquid contained in the processing chamber 30may flow to one or more of these optional components before flowingthrough the filter assembly 36. For example, liquid may flow through thepump 34 before it is passed to the filter assembly 36. Or, liquid maypass through the pressure sensor 39 before passing through the filterassembly to obtain a pre-filter liquid pressure in the system. Incertain situations, one or more of these components may also be providedas an element of the processing chamber 30, such as the vent 32 asillustrated in FIG. 6. In the illustrated embodiment, the pressuresensor 39 is in line to determine the pressure of the regenerative cellcomposition which is generated by the pump 34 as it enters the filteringchamber of the filter assembly 36. This construction can facilitatemonitoring of the trans-membrane pressure across the filter membrane.Additional saline or other buffer and washing solution can be added tothe regenerative cell composition to assist in the removal of unwantedproteins as the composition is being filtered through the filterassembly 36. This repeated washing can be performed multiple times toenhance the purity of the regenerative cells. In certain embodiments,the saline can be added at any step as deemed necessary to enhancefiltration.

In one specific embodiment, which is provided by way of example and notlimitation, the unwanted proteins and saline or other washing solutionis removed in the following manner. The composition with theregenerative cells, as well as collagen and connective tissue particlesor fragments, adipocytes, and collagenase, is cycled through a series offilters until a minimum volume is reached. The minimum volume is afunction of the total hold up volume of the system and somepredetermined constant. The hold up volume is the volume of liquid whichis contained in the tubing and conduits if all of the processingchambers are empty. In one embodiment, the minimum volume is 15 ml. Whenthe minimum volume is reached, a predetermined volume of washingsolution is introduced into the system to be mixed with the regenerativecell composition. This mixture of washing solution and the regenerativecell composition is then cycled through the filters until the minimumvolume is reached again. This cycle can be repeated multiple times toenhance the purity of the regenerative cells, or in other words, toincrease the ratio of regenerative cells in the composition to the othermaterials in the composition. See FIGS. 10 and 11.

After it has been determined that the regenerative cell composition hasbeen cleansed of unwanted proteins and concentrated sufficiently (inexemplary embodiments, minimum concentrations within a range of about1×10⁵ to about 1×10⁷ cells/ml can be used and, in a preferred embodimentthe minimum concentration can be about 1×10⁷ cells/ml), an outputchamber 50, such as an output bag, may be connected to an outlet port ofthe processing chamber 30 and/or the filter assembly 36, depending onthe specific embodiment. A vent, such as the vent 32, may then be openedto facilitate the output of the concentrated regenerative cells. In oneimplementation, this determination of when a minimum concentration hasbeen reached is made empirically after experiments have been run andprogrammed into the electronic controls of the device. The determinationcan be an input into the process of what is desired to yield, i.e., howmany stem/progenitor cells are desired, or range of cell concentration.Based on scientific data, a predefined amount of adipose tissue needs tobe obtained and placed into the system to achieve the desired output.With the vent 32 open, a pump, such as the pump 34, can function totransfer the concentrated regenerative cells into the output bag. In oneembodiment, the output bag 50 is similar to an empty blood bag which hasa tube with a fitting on one end. In a sterile fashion, the fitting onthe output bag may be attached to the outlet port, and the concentratedregenerative cells may be transferred to the output bag.

As illustrated in FIGS. 1-3, a vacuum pump 26 may be provided in thesystem 10 to change the pressure in the system, among other things. Forexample, the vacuum pump 26 may be coupled to the collection chamber 20via a conduit, such as conduit 12 b, to cause a decrease in pressurewithin the collection chamber 20. Vacuum pump 26 may also be coupled tothe processing chamber 30 by way of a conduit, such as conduit 12 g.Regarding the operation of vacuum pump 26 in connection with pump 34,two separate vacuum pumps or sources may be implemented, or a single onemay be implemented by using valves which direct the vacuum pull to thedifferent conduits that need it at specific points in the process. Inaddition, vacuum pump 26 may be coupled to the waste container 40 via aconduit, such as conduit 12 f.

With reference to FIGS. 10 and 11, the pressure generated by the vacuumpump 26 can be used to direct the flow of fluids, including theregenerative cells, through the conduits 12. This pressure can besupplied in multiple directions, for example, by automatically ormanually controlling the position of one or more valves 14 in the system10. The system 10 can be made to function properly with the use ofpositive pressure or through the use of negative pressure, orcombinations thereof. For instance, the regenerative cells can be pulledthrough the first and second filters described above into a soft sidedcontainer which is connected to the third filter. The soft-sidedcontainer can be in line (serial) connected ahead of the third filter.The final output chamber may be a soft sided container which is on theother side (e.g., the downstream side) of the third filter. In thisembodiment, pressure is used to move the regenerative cells from onesoft sided container to a second soft sided container through thefilter.

In another embodiment of the system 10, the filtration of the stem cellsand/or adipose derived progenitor cells may be accomplished using acombination of percolative filtration and sedimentation. For example,such a system uses saline that is passed through a tissue regenerativecell composition (e.g., the composition containing the stem cells and/oradipose derived progenitor cells) and then through a filter. Some of thevariables which are associated with percolative filtration of cells froma regenerative cell composition include, but are not limited to, poresize of the filter media, pore geometry or shape, surface area of thefilter, flow direction of the regenerative cell composition beingfiltered, flow rate of the infused saline, trans-membrane pressure,dilution of the cell population, cell size and viability.

In one embodiment of the system 10, the processing chamber 30 uses afilter assembly 36 which implements percolative filtration andsedimentation to separate and concentrate the regenerative cells. By wayof example, and not by way of limitation, the processing chamber 30 isdefined as a generally cylindrical body having a sidewall 30 a, a topsurface 30 b, and a bottom surface 30 c, as shown in FIG. 6. A sterilevent 32 is provided in the top surface 30 b.

In the embodiment of FIG. 6, the processing chamber 30 is illustrated asincluding a filter assembly 36, which includes two filters, such aslarge pore filter 36 a, and small pore filter 36 b. The pore sizes ofthe filters 36 a and 36 b typically are in a range between about 0.05microns and about 10 microns. The large pore filter 36 a may comprisepores with a diameter of about 5 μm, and the small pore filter 36 b maycomprise pores with a diameter of about 1-3 μm. In one embodiment, thefilters have a surface area of about 785 mm². Filters 36 a and 36 bdivide an interior of the processing chamber 30 to include a firstchamber 37 a, a second chamber 37 b, and a third chamber 37 c. As shownin FIG. 6, first chamber 37 a is located between second chamber 37 b andthird chamber 37 c. In addition, first chamber 37 a is shown as beingthe region of the processing chamber 30 having an inlet port 31 a and anoutlet port 31 b. The illustrated processing chamber 30 includes aplurality of ports providing communication paths from an exterior of theprocessing chamber 30 to the interior of the processing chamber 30, suchas ports 31 a, 31 b, and 31 c. The ports 31 a, 31 b, and 31 c, areillustrated as being disposed in the sidewall 30 a of a body of theprocessing chamber 30. However, the ports 31 a, 31 b, and 31 c could bepositioned in other regions, as well. Port 31 a is illustrated as asample inlet port, which is constructed to be coupled to a conduit sothat a composition containing regenerative cells can be passed into theinterior of the processing chamber 30. Port 31 b is illustrated as anoutlet port constructed to be coupled to a conduit so that the separatedand concentrated cells may be removed from the interior of theprocessing chamber 30. Port 31 c is illustrated as an inlet portconstructed to be coupled to a conduit for delivery of a fresh washingsolution, such as saline into the interior of the processing chamber 30.

In use, the regenerative cells may be introduced into the centralchamber 37 a via inlet port 31 a. Saline or other buffer is introducedinto the bottom chamber 37 b through inlet port 31 c. The saline may bedirected through the regenerative cell composition in chamber 37 a at arate of about 10 ml/min. The flow rate of the saline is such that itcounteracts the force of gravity. The flow of saline gives the cells inthe chamber the ability to separate based on the density of the cells.Typically, as the saline is forced up through the composition the largercells in the composition will settle to the bottom of the centralchamber 37 a, and the smaller cells and proteins will be carried awaythrough the second filter 36 b into the top chamber 37 c. This filteringis accomplished by adjusting the flow rate of the saline such that thelarger cells are rolled in place which allows the smaller particles tobe liberated and carried off with the saline. The sterile vent 32 isincluded in the chamber 30 to ensure that the correct pressure gradientis maintained in the three chambers within the processing unit. Theupper chamber 37 c can comprise an absorbent media 33. The purpose ofthe absorbent media is to trap the unwanted proteins in the solution toensure that they do not cross the filter media back into the processingsolution, if, for example, the saline flow rate decreases. An absorbentmedia can be a type of filter material that is absorbent, or attractsmaterials or components to be filtered out. An outflow port can be addedabove the top filter to help draw off the waste. Another embodiment ofthis may be to apply a gentle vacuum from the top to help pull offwaste. Absorbent media can be implemented when, as in the illustratedembodiment, the flow rates are relatively small. Excess saline andproteins are then carried away to a waste container.

When the larger cells, (e.g., the adipose derived stem cells and/orprogenitor cells) have been sufficiently separated from smaller cellsand proteins, the composition containing the separated cells may beconcentrated, as discussed herein. The composition may be furtherconcentrated after it has been removed from chamber 37 a through outletport 31 b, or while it is in the chamber 37 a. In one embodiment, theconcentration of cells in the composition is increased in the followingmanner. After the cells have been sufficiently separated the filters,such as filters 36 a and 36 b, may be moved towards each other. Thismovement has the effect of reducing the volume between the two filters(e.g., the volume of chamber 37 a). A vibrating member may also beprovided in connection with the processing chamber 30 to facilitateconcentrating of the cells in the composition. In one embodiment, thevibrating member may be coupled to the filter 36 b (e.g., the small porefilter). Vibrating can reduce an incidence of cells becoming trapped inthe filters. The reduction in volume of the composition allows theexcess saline to be removed as waste and the cells to be concentrated ina smaller volume.

In another embodiment, the concentration of the regenerative cells isaccomplished in the following manner. After the cells have beensufficiently separated, the regenerative cell composition can betransferred to another chamber (not shown) which uses gravity to filterout the excess saline. In a preferred embodiment, the sedimentation canoccur at the same time as the percolation. This sedimentation may beaccomplished by introducing the composition on top of a filter which hasa pore size ranging from about 10 kD to about 2 microns. In oneembodiment, a suitable filter has a pore size of about 1 micron. Theforce of gravity will allow the saline and smaller particles to bepassed through the filter while preventing the cells in the compositionto flow through the filter. After the desired concentration of cells hasbeen obtained, and after the filtered smaller particles have beenremoved from below the filter, the regenerative cell composition may beagitated to remove the cells from the filter and, subsequently, theconcentrated regenerative cells may be transferred to the output bag.The smaller particles can be drawn off as waste through an outlet.

In a particular embodiment, the regenerative cell composition from thecollection chamber 20 is transported to the processing chamber 30wherein the composition can be centrifuged to separate and concentrateregenerative cells. Centrifugation principles are well know in the artand will be not be repeated herein in the interest of brevity. Standard,art-recognized centrifugation devices, components and parameters areutilized herein. An exemplary processing chamber for use as part of acentrifuge device is shown in FIGS. 7 and 8. Typically, a centrifugedevice causes a centrifuge chamber (such as the one shown in FIG. 7) tospin around an axis to thereby increasing the force on the cells in thesolution to be greater than gravity. The denser or heavier materials inthe solution typically settle to one end of the centrifuge chamber,i.e., an output chamber 50 of FIG. 7, to form a regenerative cellpellet. The pellet may then be re-suspended to obtain a solution with adesired concentration of cells and/or a desired volume of cells andmedium. The processing chamber shown in FIG. 7 is constructed toseparate and concentrate cells using both centrifugal and gravitationalforces. Specifically, during centrifugation, centrifugal force directsthe denser components of the regenerative cell composition, e.g., theregenerative cells, towards the outermost ends of the centrifugechamber. As the centrifuge chamber slows down and eventually stops,gravitational force helps the regenerative cells to remain in theoutermost ends of the centrifuge chamber and form a cell pellet.Accordingly, the unwanted components of the regenerative cellcomposition, i.e., the waste, can be removed without disturbing the cellpellet.

In yet another embodiment of the invention, the processing chamber maybe comprised of a cell concentrator in the form of a spinning membranefilter. In a further embodiment of the centrifugation process,centrifugal elutriation may also be applied. In this embodiment, thecells may be separated based on the individual cell sedimentation ratesuch that the directional (e.g., outward) force applied bycentrifugation causes cells and solutes to sediment at different rates.In elutriation, the sedimentation rate of the target cell population isopposed by an opposite (e.g., inward) flow rate applied by pumpingsolution in the opposite direction to the centrifugal force. Thecounterflow is adjusted so that the cells and particles within thesolution are separated. Elutriation has been applied in many instancesof cell separation (Inoue, Carsten et al. 1981; Hayner, Braun et al.1984; Noga 1999) and the principles and practices used to optimize flowand centrifugal parameters can be applied herein in light of the presentdisclosure by one skilled in the art.

FIG. 9 illustrates principles associated with an elutriationimplementation in accordance with the present invention. The elutriationembodiment can be similar to a centrifugation implementation to theextent that a force is applied to the solution using a spinning rotor.Some of the variables which are associated with the presently embodiedelutriation separation include, but are not limited to, the size andshape of the spinning chamber, the diameter of the rotor, the speed ofthe rotor, the diameter of the counter flow tubing, the flow rate of thecounter flow, as well as the size and density of the particles and cellswhich are to be removed from solution. As in centrifugation, theregenerative cells can be separated based on individual cell densities.

In one embodiment the regenerative cell composition, e.g., the solutioncontaining the regenerative cells and the collagenase, is introducedinto a chamber of a spinning rotor, as shown in FIG. 9.1. After thesolution is added to the chamber additional saline is added to thechamber at a predetermined flow rate. The flow rate of the saline can bepredetermined as a function of the speed of the rotor, the celldiameter, and the chamber constant which has been establishedempirically. The flow rate will be controlled for example with a devicesimilar to an IV pump. A purpose of the additional saline is to providea condition inside the rotor chamber where the larger particles willmove to one side of the chamber and the smaller particles will move tothe other, as illustrated in FIG. 9.2. The flow is adjusted so that, inthis application, the smaller particles will exit the chamber and moveto a waste container, as shown in FIG. 9.3. This movement results in thesolution in the rotor chamber having a substantially homogenouspopulation of cells, such as stem cells. After it has been determinedthat the stem cells have been separated from the rest of the items inthe solution (with unwanted proteins and free lipids having been removedfrom the chamber), the counter flow is stopped. The cells inside thechamber will then form a concentrated pellet on the outside wall of thechamber. The counter flow is reversed and the cell pellet is transferredto the output bag.

As previously set forth herein, the processing chamber 30 or the outputchamber 50 may include one or more ports, e.g., ports 51 or 52. One ormore of these ports may be designed to transport the regenerative cellsobtained using any combination of methods described above, or a portionthereof, via conduits to other surgical devices, cell culturing devices,cell marinading devices, gene therapy devices or purification devices.These ports may also be designed to transport the regenerative cells viaconduits to additional chambers or containers within the system or aspart of another system for the same purposes described above. The portsand conduits may be also be used to add one or more additives, e.g.,growth factors, re-suspension fluids, cell culture reagents, cellexpansion reagents, cell preservation reagents or cell modificationreagents including agents that transfer genes to the cells. The portsand conduits may also be used to transport the regenerative cells toother targets such as implant materials (e.g., scaffolds or bonefragments) as well as other surgical implants and devices.

Further processing of the cells may also be initiated by reconfiguringthe interconnections of the disposable sets of the existing system,re-programming the processing device of the existing system, byproviding different or additional containers and/or chambers for theexisting system, by transporting the cells to a one or more additionalsystems or devices and/or any combinations thereof. For example, thesystem can be reconfigured by any of the means described above such thatthe regenerative cells obtained using the system may be subject to oneor more of the following: cell expansion (of one or more regenerativecell types) and cell maintenance (including cell sheet rinsing and mediachanging); sub-culturing; cell seeding; transient transfection(including seeding of transfected cells from bulk supply); harvesting(including enzymatic, non-enzymatic harvesting and harvesting bymechanical scraping); measuring cell viability; cell plating (e.g., onmicrotiter plates, including picking cells from individual wells forexpansion, expansion of cells into fresh wells); high throughputscreening; cell therapy applications; gene therapy applications; tissueengineering applications; therapeutic protein applications; viralvaccine applications; harvest of regenerative cells or supernatant forbanking or screening, measurement of cell growth, lysis, inoculation,infection or induction; generation of cells lines (including hybridomacells); culture of cells for permeability studies; cells for RNAi andviral resistance studies; cells for knock-out and transgenic animalstudies; affinity purification studies; structural biology applications;assay development and protein engineering applications.

For example, if expansion of a regenerative cell population is requiredfor a particular application, an approach using culture conditions topreferentially expand the population while other populations are eithermaintained (and thereby reduced by dilution with the growing selectedcells) or lost due to absence of required growth conditions could beused. Sekiya et al have described conditions which might be employed inthis regard for bone marrow-derived stem cells (Sekiya et al., 2002).This approach (with or without differential adherence to the tissueculture plastic) could be applied to a further embodiment of thisinvention. In this embodiment the final regenerative cell pellet isremoved from the output chamber and placed into a second systemproviding the cell culture component. This could be in the form of aconventional laboratory tissue culture incubator or a Bioreactor-styledevice such as that described by Tsao et al., U.S. Pat. No. 6,001,642,or by Armstrong et al., U.S. Pat. No. 6,238,908. In an alternativeembodiment, the cell expansion or cell culture component could be addedto the existing system, e.g., into the output chamber, allowing forshort-term adherence and/or cell culture of the adipose derived cellpopulations. This alternate embodiment would permit integration of thecell culture and/or cell expansion component to the system and removethe need for removing the cells from this system and placement withinanother.

During the processing, one or more additives may be added to or providedwith the various chambers or containers as needed to enhance theresults. These additives may also be provided as part of another systemassociated with the existing system or separate from the existingsystem. For example, in certain embodiments, the additives are added orprovided without the need for removing the regenerative cells from thesystem. In other embodiments, the additives are added or provided byconnecting a new container or chamber comprising the additives into anunused port of the system in a sterile manner. In yet other embodiments,the additives are added or provided in a second system or device that isnot connected to the system of the present invention. Some examples ofadditives include agents that optimize washing and disaggregation,additives that enhance the viability of the active cell populationduring processing, anti-microbial agents (e.g., antibiotics), additivesthat lyse adipocytes and/or red blood cells, or additives that enrichfor cell populations of interest (by differential adherence to solidphase moieties or to otherwise promote the substantial reduction orenrichment of cell populations) as described herein.

For example, to obtain a homogenous regenerative cell population, anysuitable method for separating and concentrating the particularregenerative cell type may be employed, such as the use of cell-specificantibodies that recognize and bind antigens present on, for example,stem cells or progenitor cells, e.g., endothelial precursor cells. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof. Intracellular markers such as enzymes may also be used inselection using molecules which fluoresce when acted upon by specificenzymes. In addition, a solid phase material with adhesive propertiesselected to allow for differential adherence and/or elution of aparticular population of regenerative cells within the final cell pelletcould be inserted into the output chamber of the system.

An alternate embodiment of this differential adherence approach wouldinclude use of antibodies and/or combinations of antibodies recognizingsurface molecules differentially expressed on target regenerative cellsand unwanted cells. Selection on the basis of expression of specificcell surface markers (or combinations thereof) is another commonlyapplied technique in which antibodies are attached (directly orindirectly) to a solid phase support structure (Geiselhart et al., 1996;Formanek et al., 1998; Graepler et al., 1998; Kobari et al., 2001; Mohret al., 2001).

In another embodiment the cell pellet could be re-suspended, layeredover (or under) a fluid material formed into a continuous ordiscontinuous density gradient and placed in a centrifuge for separationof cell populations on the basis of cell density. In a similarembodiment continuous flow approaches such as apheresis (Smith, 1997),and elutriation (with or without counter-current) (Lasch et al., 2000)(Ito and Shinomiya, 2001) may also be employed.

Other examples of additives may include additional biological orstructural components, such as cell differentiation factors, growthpromoters, immunosuppressive agents, medical devices, or anycombinations thereof, as discussed herein. For example, other cells,tissue, tissue fragments, growth factors such as VEGF and other knownangiogenic or arteriogenic growth factors, biologically active or inertcompounds, resorbable scaffolds, or other additives intended to enhancethe delivery, efficacy, tolerability, or function of the population ofregenerative cells may be added. The regenerative cell population mayalso be modified by insertion of DNA or by placement in a cell culturesystem (as described herein or known in the art) in such a way as tochange, enhance, or supplement the function of the regenerative cellsfor derivation of a structural or therapeutic purpose. For example, genetransfer techniques for stem cells are known by persons of ordinaryskill in the art, as disclosed in (Morizono et al., 2003; Mosca et al.,2000), and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in (Walther and Stein, 2000) and (Athanasopoulos et al.,2000). Non-viral based techniques may also be performed as disclosed in(Muramatsu et al., 1998). A gene encoding one or more cellulardifferentiating factors, e.g., a growth factor(s) or a cytokine(s),could also be added. Examples of various cell differentiation agents aredisclosed in (Gimble et al., 1995; Lennon et al., 1995; Majumdar et al.,1998; Caplan and Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger etal., 1999; Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001;Zuk et al., 2001). Genes encoding anti-apoptotic factors or agents couldalso be added. Addition of the gene (or combination of genes) could beby any technology known in the art including but not limited toadenoviral transduction, “gene guns,” liposome-mediated transduction,and retrovirus or lentivirus-mediated transduction, plasmid,adeno-associated virus. These regenerative cells could then be implantedalong with a carrier material bearing gene delivery vehicle capable ofreleasing and/or presenting genes to the cells over time such thattransduction can continue or be initiated in situ.

When the cells and/or tissue containing the cells are administered to apatient other than the patient from whom the cells and/or tissue wereobtained, one or more immunosuppressive agents may be administered tothe patient receiving the cells and/or tissue to reduce, and preferablyprevent, rejection of the transplant. As used herein, the term“immunosuppressive drug or agent” is intended to include pharmaceuticalagents which inhibit or interfere with normal immune function. Examplesof immunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell costimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No.20020182211. A preferred immunosuppressive agent is cyclosporine A.Other examples include myophenylate mofetil, rapamicin, andanti-thymocyte globulin. In one embodiment, the immunosuppressive drugis administered with at least one other therapeutic agent. Theimmunosuppressive drug is administered in a formulation which iscompatible with the route of administration and is administered to asubject at a dosage sufficient to achieve the desired therapeuticeffect. In another embodiment, the immunosuppressive drug isadministered transiently for a sufficient time to induce tolerance tothe regenerative cells of the invention.

In these embodiments, the regenerative cells may be contacted, combined,mixed or added to the additives through any art recognized manner,including devices such as the agitation devices and associated methodsdescribed herein. For example, rocking, inversion, compression pulsed ormoving rollers may be used.

In another aspect, the cell population could be placed into therecipient and surrounded by a resorbable plastic sheath or othermaterials and related components such as those manufactured by MacroPoreBiosurgery, Inc. (see e.g., U.S. Pat. Nos. 6,269,716; 5,919,234;6,673,362; 6,635,064; 6,653,146; 6,391,059; 6,343,531; 6,280,473).

In all of the foregoing embodiments, at least a portion of the separatedand concentrated regenerative cells may be cryopreserved, as describedin U.S. patent application Ser. No. 10/242,094, entitled PRESERVATION OFNON EMBRYONIC CELLS FROM NON HEMATOPOIETIC TISSUES, filed Sep. 12, 2002,which claims the benefit of U.S. Provisional Patent Application60/322,070 filed Sep. 14, 2001, which is commonly assigned, and thecontents of which in their entireties are expressly incorporated hereinby reference.

As set forth herein, the regenerative cells obtained using the systemsand methods of the present invention contain a variety of different celltypes including regenerative cells. Typically the range of differentcell types will be as shown in Table II. However, these ranges will bedependent on factors including, but not limited to, donor age, medicalcondition, the manner in which the tissue was obtained. For example,tissue removed by lipectomy (excision) will contain less blood and thusthe final product will have fewer residual mature blood cells such aslymphocytes. Similarly, application of supplemental procedures such ascountercurrent elutriation, density centrifugation, differentialadherence, and other procedures outlined herein would, by design andintent, considerably change these ranges.

TABLE II Characterization of Cell Populations Within Regenerative CellsSurface Marker Marker Comment Frequency Range (%) CD45 Nucleated bloodcells 13-56 CD31 Endothelial cell  6-32 ABCG2 Stem cell-associatedmarker 1-8 CD184 Chemokine receptor  3-16 CD105 Endoglin 0-2 CD71Transferrin receptor  0-17 CD34 Endothelial cells and stem cells 17-72CD29 β1 integrin  9-21

In addition to the foregoing, there are many post-wash methods that maybe applied for further purifying the active cell population. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof. In all embodiments of positive and negative selection describedherein, the selection materials used will be endotoxin free, will notleach into the active cell population, and will not activate anyphagocytic cell types present in the active cell population, such aspre-adipocytes, tissue macrophages, or monocytes. Further, the systemsin which such procedures are performed shall be closed or functionallyclosed sterile and free of contaminants. Such systems may be fullyintegrated with the systems used earlier in manufacture or may beseparate modules joined by creation of a sterile fluid path or bysterile or aseptic transfer of material from the earlier systems asdescribed herein.

At the end of processing, the regenerative cells may be manuallyretrieved from the output chamber. The cells may be loaded into adelivery device, such as a syringe, for placement into the recipient byeither, subcutaneous, intramuscular, or other technique allowingdelivery of the cells to the target site within the patient. In otherwords, cells may be placed into the patient by any means known topersons of ordinary skill in the art. Preferred embodiments includeplacement by needle or catheter, or by direct surgical implantation. Inother embodiments, the cells may be automatically transported to anoutput chamber which may be in the form of a container, syringe orcatheter etc., which may be used to place the cells in the patient. Thecontainer may also be used to store the cells for later use or forcryopreservation. All retrieval methods are performed in a sterilemanner. In the embodiment of surgical implantation, the cells could beapplied in association with additives such as a preformed matrix orscaffold as described herein.

In preferred embodiments of the invention (e.g., the embodiment shown inFIG. 4), the system is automated. In another embodiment, the system hasboth automated and manual components. The system may be comprised of oneor more disposable components connected to or mounted on a re-usablehardware component or module. The automated systems of the inventionprovide screen displays (see FIG. 16) that prompt proper operation ofthe system. The automated systems may also provide a screen thatprovides status of the procedure and/or the step by step instructions asto the proper setup of the disposable components of the system. Thescreen may also indicate problems or failures in the system if theyoccur and provide “troubleshooting” guidance if appropriate. In oneembodiment, the screen is a user interface screen that allows the userto input parameters into the system through, e.g., a touch screen.

The partial and fully automated systems may include a processing device(e.g., microprocessor or personal computer) and associated softwareprograms that provide the control logic for the system to operate and toautomate one or more steps of the process based on user input. Incertain embodiments, one or more aspects of the system may beuser-programmable via software residing in the processing device. Theprocessing device may have one or more pre-programmed software programsin Read Only Memory (ROM). For example, the processing device may havepre-programmed software tailored for processing blood, another programfor processing adipose tissue to obtain small volumes of regenerativecells and another program for processing adipose tissue to obtain largervolumes of regenerative cells. The processing device may also havepre-programmed software which provides the user with appropriateparameters to optimize the process based on the user's input of relevantinformation such as the amount of regenerative cells required, the typeof tissue being processed, the type of post-processing manipulationrequired, the type of therapeutic application, etc.

The software may also allow automation of steps such as controlling theingress and egress of fluids and tissues along particular tubing pathsby controlling pumps and valves of the system; controlling the propersequence and/or direction of activation; detecting blockages withpressure sensors; mixing mechanisms, measuring the amount of tissueand/or fluid to be moved along a particular pathway using volumetricmechanisms; maintaining temperatures of the various components usingheat control devices; and integrating the separation and concentrationprocess with timing and software mechanisms. The processing device canalso control centrifuge speeds based on the tissue type being processedand/or the cell population or sub-population being harvested, and thetypes of procedures to be performed (e.g., tissue enhancement usingadipose tissue augmented with regenerative cells, or processing of cellsfor bone repair applications using regenerative cell coated bonegrafts). The processing device may also include standard parallel orserial ports or other means of communicating with other computers ornetworks. Accordingly, the processing device can be a stand alone unitor be associated one or more additional devices for the furtherprocessing methods described herein.

The software may allow for automated collection of “run data” including,for example, the lot numbers of disposable components, temperature andvolume measurements, tissue volume and cell number parameters, dose ofenzyme applied, incubation time, operator identity, date and time,patient identity, etc. In a preferred embodiment of the device acharacter recognition system, such as a bar code reading system would beintegrated to permit data entry of these variables (for exampledisposable set lot number and expiration date, lot number and expirationdate of the Collagenase, patient/sample identifiers, etc.) into theprocessing device as part of documentation of processing. This wouldreduce the opportunity for data entry errors. Such a bar code readingsystem could be easily incorporated into the processing device using aUSB or other interface port and system known to the art. In this way thedevice would provide integrated control of the data entry anddocumentation of the process. A print-out report of these parameterswould be part of the user-defined parameters of a programmed operationof the system. Naturally this would require integration of a printercomponent (hardware and driver) or printer driver in software plus aninterface output connector for a printer (e.g., a USB port) in thehardware of the device.

In certain embodiments, the system is a fully automated system. Forexample, the user may initially select the amount of tissue to beprocessed, attach the system to the patient and the system mayautomatically aspirate the required tissue and separate and concentrateregenerative cells in an uninterrupted sequence without further userinput. The user may also input the amount of regenerative cells requiredand allow the system to aspirate the requisite amount of tissue andprocess the tissue. A fully automated system also includes a systemwhich is capable of being reconfigured based on a number of (e.g., twoor more) user input parameters, e.g., number of wash cycles, speed ofcentrifugation etc. The system can also be run in semi-automatic modeduring which the system goes through certain steps without userintervention but requires user intervention before certain processes canoccur. In other embodiments, the system is a single integrated systemthat displays instructions to guide the user to perform predeterminedoperations at predetermined times. For example, the processing devicemay prompt users through the steps necessary for proper insertion oftubing, chambers and other components of the system. Accordingly, theuser can ensure that the proper sequence of operations is beingperformed. Such a system can additionally require confirmation of eachoperational step by the user to prevent inadvertent activation ortermination of steps in the process. In a further embodiment, the systemmay initiate automated testing to confirm correct insertion of tubing,chambers, absence of blockages etc. In yet another embodiment, thesystem of the present invention is capable of being programmed toperform multiple separation and concentration processes throughautomated control of tissue flow through the system. This feature may beimportant, for example, during surgery on a patient where tissue thatwould otherwise be lost is collected into the system, and regenerativecells from the tissue are separated and concentrated and returned to thepatient.

As set forth above, components of the system may be disposable (referredto herein as “disposable set(s)”), such that portions of the system canbe disposed of after a single use. This implementation can help ensurethat any surface which comes in contact with the patient's tissue willbe disposed of properly after being used. An exemplary disposable set isillustrated in FIG. 13. In a preferred embodiment, the disposablecomponents of the system are pre-sterilized and packaged so as to beusable “off the shelf” that are easy to use and easy to load and thateliminate the need for many tubing connections and complex routing oftubing connections. Such disposable components are relativelyinexpensive to manufacture, and therefore, do not create a substantialexpense due to their disposal. In one embodiment, the disposable system(referred to interchangeably herein as “disposable set(s)”) comprises,consists essentially of, or consists of, the collection chamber 20, theprocessing chamber 30, the waste chamber 40, the output chamber 50, thefilter assemblies 36, the sample bag 60 and the associated conduits 12or tubing. In preferred embodiments of the disposable sets of thesystem, the collection chamber 20 and the processing chamber 30 areconnected by way of conduits 12 that are housed in a rigid frame. Therotating seal network (FIGS. 7 & 8) of a processing chamber 30 may alsobe housed in the same rigid frame. In another preferred embodiment, thevarious chambers and containers of the disposable set are comprised ofthe necessary interfaces that are capable of communicating with theprocessing device of the system such that the pumps, valves, sensors andother devices that automate the system are appropriately activated orde-activated as needed without user intervention. The interfaces alsoreduce the time and expertise required to set up the system and alsoreduce errors by indicating how to properly set up the system andalerting the user in the event of an erroneous setup.

Most of the disposable sets of the invention will have many commonelements. However, the ordinarily skilled artisan will recognize thatdifferent applications of the system may require additional componentswhich may be part of the disposable sets. Accordingly, the disposablesets may further comprise one or more needles or syringes suitable forobtaining adipose or other tissue from the patient and returningregenerative cells to the patient. The type number and variety of theneedles and syringes included will depend on the type and amount oftissue being processed. The disposable sets may further comprise one ormore rigid or flexible containers to hold washing fluids and otherprocessing reagents used in the system. For example, the disposable setsmay comprise containers to hold saline, enzymes and any other treatmentor replacement fluids required for the procedure. In addition, suitablewashing solutions, re-suspension fluids, additives, agents or transplantmaterials may be provided with the disposable sets for use inconjunction with the systems and methods of the invention.

Any combination of system components, equipment or supplies describedherein or otherwise required to practice the invention may be providedin the form of a kit. For example, a kit of the invention may include,e.g., the optimal length and gage needle for the syringe basedliposuction and sterile syringes which contain the preferred filtermedia which allows for the processing of small volumes of tissue. Otherexemplary equipment and supplies which may be used with the inventionand may also be included with the kits of the invention are listed inTables III and IV.

Table III below identifies examples of supplies that can be used in toobtain adipose derived regenerative cell in accordance with the systemsand methods of the present invention:

TABLE III Description Vendor Quantity Note 10 ml syringe Becton- asreq'd Optional, used for lipo- Dickinson suction 14 GA blunt tip asreq'd Optional, used for lipo- needle suction Single Blood Baxter 1 Maincell processing bag; Pack (600 ml) Fenwal bag has spike adaptor on lineand two free spike ports Transfer pack Baxter 1 Quad bag set withcoupler Fenwal (150 ml) Transfer pack Baxter 1 Waste bag with couplerFenwal (1 L) Sample Site Baxter 2 Coupler Fenwal 0.9% saline (for Baxter1 injection) Fenwal 14 GA sharp Monoject as req'd For adding liposuctionneedle tissue to bag 20 GA sharp Monoject 3 For adding collagenase andneedle removing PLA cells 0.2 μm Sterflip Millipore 1 For filteringcollagenase filter Teruflex Aluminium Terumo 4 ME*ACS121 for temporarysealing clips tube sealing Povidone Iodine Triadine as req'd 10-3201prep pad Liberase H1 Roche See Procedure Note 1 Collagenase TSCD wafersTerumo 2 1SC*W017 for use with TSCD Sterile Tubing Welder

Table IV, below, identifies equipment that may be used with the systemsand methods disclosed herein.

TABLE IV Description Vendor Quantity Note Sorvall Legend T Easy Fisher 175-004-367 Set Centrifuge Scientific Rotor Kendro/ 1 TTH-750 rotorSorvall Rotor buckets Kenro/ 4 75006441 round Sorvall buckets Adaptorfor 150 ml bags Kendro/ 4 00511 Sorvall Plasma Expressor Baxter Fenwal 14R4414 Tube Sealer Sebra 1 Model 1060 TSCD Sterile Tubing Terumo 13ME*SC201AD Welder LabLine Thermal Rocker LabLine 1 4637 ‘Disposable’plastic Davron 3 hemostat-style clamp Balance Bags Sets 2 Water-filledbags used to balance centrifuge Biohazard Sharps 1 Chamber BiohazardWaste 1 Chamber

The re-usable component of the system comprises, consists essentiallyof, or consists of the agitation mechanism for the collection chamber,the pump, and assorted sensors which activate valves and pump controls,the centrifuge motor, the rotating frame of the centrifuge motor, theuser interface screen and USB ports, an interlocking or docking deviceor configuration to connect the disposable set such that the disposableset is securely attached to and interface with the re-usable hardwarecomponent and other associated devices. An exemplary re-usable componentis illustrated in FIG. 14. In preferred embodiments, the re-usablecomponent includes a means for separating and concentrating theregenerative cells from the regenerative cell composition, e.g., arotating centrifuge. In this embodiment, the re-usable component isdesigned connect to and interface with a portion of the processingchamber (comprising a centrifuge chamber) of the disposable set as shownin FIG. 15A. It is understood that the means for separating andconcentrating regenerative cells in the re-usable component is notlimited to a rotating centrifuge but may also include any otherconfiguration described herein, including a spinning membrane filter.The re-usable component may also house the processing device describedherein which contains pre-programmed software for carrying out severaldifferent tissue processing procedures and selectively activating thevarious pumps and valves of the system accordingly. The processor mayalso include data storage capability for storing donor/patientinformation, processing or collection information and other data forlater downloading or compilation. The re-usable component may be usedwith a variety of disposable sets. The disposable set is connected tothe re-usable component through, e.g., an interlocking device orconfiguration to connect the disposable set such that the disposable setis securely attached to and interfaces with the re-usable hardwarecomponent in a manner that the processing device present on there-usable component can control, i.e., send and receive signals to andfrom the various components of the disposable set as well as variouscomponents of the re-usable component and other associated devices andsystems.

In one embodiment, a disposable set for use in the system is comprisedof a collection chamber 20 which can accommodate about 800 mL of tissue;a processing chamber 30 which can process the regenerative cellcomposition generated by about 800 mL of tissue washed and digested inthe collection chamber 20; an output chamber 50 which can accommodate atleast 0.5 mL of regenerative cells; and a waster container 40 which canaccommodate about 10 L of waste. In this embodiment, the hardware deviceis no larger than 24″L×18″W×36″H. Alternative dimensions of the variouscomponents of the disposable sets as well as the hardware device may beconstructed as needed and are intended to be encompassed by the presentinvention without limitation.

The disposable components of the system are easy to place on the device.An illustration of a disposable set utilized assembled together with acorresponding re-usable component is illustrated in FIG. 15A. The systemis preferably designed such that it can detect an improperly loadeddisposable component. For example, the components of each disposable setmay have color-guided marks to properly align and insert the tubing,chambers etc. into appropriate places in the system. In additionalembodiments, the system disclosed herein is a portable unit. Forexample, the portable unit may be able to be moved from one locationwhere adipose tissue harvesting has occurred, to another location foradipose tissue harvesting. In certain implementations, the portable unitis suitable for harvesting and processing of adipose tissue by apatient's bedside. Thus, a portable unit may be part of a system whichcan be moved from patient to patient. Accordingly, the portable unit maybe on wheels which lock in place and, thus, can be easily placed andused in a convenient location in a stable and secure position throughoutthe procedure. In other embodiments, the portable unit is designed forset-up and operation on a flat surface such as a table top. The portableunit may also be enclosed in a housing unit. The portable unit mayfurther be comprised of hangers, hooks, labels, scales and other devicesto assist in the procedure. All of the herein described re-usablecomponents of the system such as the centrifuge, processing device,display screen may be mounted on the portable unit of the system.

Alternate manual embodiments for obtaining regenerative cells are alsowithin the scope of this invention. For example, in one embodiment,adipose tissue may be processed using any combination of the componentsof the system, equipment and/or supplies described herein.

A manual embodiment of the system of the invention may be practiced inaccordance with the following steps and information, which are providedby way of example and not by way of limitation. First, adipose tissue iscollected from a patient. A tissue retrieval line, or sampling sitecoupler, is opened and a spike is inserted into a side port of the 600ml blood bag. Approximately 10 ml of adipose tissue is collected in a 10ml syringe through the blunt cannula. The blunt cannula is replaced witha relatively sharp needle (14G). The sampling site is wiped with aniodine wipe. The adipose tissue is injected into the 600 ml bag throughthe sampling site. The syringe and needle are then discarded in a sharpschamber. These steps are repeated to place sufficient tissue into thebag. Sufficient tissue is determined on a case-by case basis based onthe clinical specifics of the patient and application.

Second, the aspirated adipose tissue is washed. A pre-warmed (37° C.)saline bag is hooked above the work surface. A blue hempostat clamp isplaced on the tubing between the 600 ml bag and the spike. The clamp isclosed to seal the tubing. The spike on the 600 ml bag is used to enterthe saline bag (in this setting use the needle on the 600 ml bag toenter the saline bag through the rubber septum, wipe the septum withiodine prior to insertion of needle). The blue clamp is released andapproximately 150 ml of saline is allowed to enter the 600 ml bag. Theblue clamp is closed when the desired volume of saline has entered the600 ml bag. The 600 ml bag is inverted 10-15 times over approximately 15seconds. A second blue clamp is applied to the tubing leading from the 3L waste bag to the spike. The spike on the 3 L bag is used to enter the600 ml bag. The 600 ml bag is hung inverted over the work surface, andis allowed to sit for approximately 1 minute. The blue clamp leading tothe 3 L bag is released. Waste fluid is allowed to flow into the 3 Lbag. The blue clamp is applied to stop the flow before tissue enters thetubing. The 600 ml bag is lowered to the work surface. These steps arerepeated two more times. If the saline waste still appears noticeablyred, a third additional cycle is indicated. A heat sealer is used toseal the tubing between the 3 L waste bag and the 600 ml bag. The sealis made at approximately the half way point on the tubing. The 3 L wastebag is removed and discarded. The 600 ml bag is returned to the worksurface.

Third, the washed adipose tissue is digested. The blue clamp on thetubing between the saline and the 600 ml bag is released to allowapproximately 150 ml of saline to enter the 600 ml bag. The samplingsite on the 600 ml bag is wiped with an iodine wipe. Collagenase isinjected through the sampling site to the 600 ml bag. The collagenase isprepared by thawing one collagenase vial in a 37° C. water bath orequivalent, other than microwaving. A 1 ml syringe with a 22G needle isinserted into the vial. The collagenase is withdrawn into the needle.The needle is removed and replaced with a 0.2 μm filter and second 22Gneedle. The collagenase is then expelled from the syringe through the0.2 μm filter and needle. Digestion of the adipose tissue is performedat a final collagenase concentration of 0.1-0.2 Wunsch units/ml. Theheating pad is placed on the rocker. During this time, the saline bag,while still attached, is set to the side of the rocker. Care is taken toensure that the tubing leading to the saline bag is positioned in such away that it does not get caught on the rocker when in motion. Theheating pad controller is set to 37° C. The 600 ml bag is placed on therocker. The rocker is set to maximum. The bag is observed to ensure thatit is stable, and is allowed to rock for approximately 1 hour (55±10mins).

Fourth, the regenerative cell composition is retrieved. The bag isremoved from the rocker. A blue clamp is applied to the closed tubingformerly leading to the waste bag. The sterile connecting device is usedto attach the quad bag set (pre-prepared according to the followinginstructions) to the tubing that was formerly attached to the waste bag.The quad pack can be seen as two linked quad packs. Identify the tubingsplitting it into two packs, fold the tubing back on itself, and slip ametal loop over the folded tubing (over both pieces of tubing). Slidethe loop down approx 0.5 inch. The crimp formed at the bend acts to sealthe tubing. Use a hemostat to partially crimp the loop closed. The loopis not crimped too tightly because the loop will need to be removedduring processing. The 600 ml bag is hung inverted over the work surfaceand is allowed to sit for approximately 3 minutes. The blue clamp ontubing leading to the quad set is released to drain the cell fraction(the layer under the yellow/orange fat layer) into the quad set. Care istaken to prevent the fat layer to enter the tubing. During this process,the tubing can be crimped manually to slow the flow as the fat layergets close to the tubing. The tubing leading to the quad bag set is thenclosed with a blue clamp, the 600 ml bag is returned to the worksurface, and the saline bag is hung. The blue clamp on the tubingbetween the saline and the 600 ml bag is released to allow approximately150 ml of saline to enter the 600 ml bag. The 600 ml bag is invertedapproximately 10-15 times over approximately 15 seconds. The 600 ml bagis then hung inverted over the work surface and is allowed to sit forabout 3-5 minutes. The blue clamp on tubing leading to the quad set isreleased, and the cell fraction (the layer under the yellow/orange fatlayer) is drained into the quad set. Care is taken to prevent the fatlayer from entering the tubing. For example, the flow can be slowed asthe fat layer gets close to the tubing by crimping the tubing manually.The tubing leading to the quad bag set is closed with a blue clamp. Thetubing leading from the quad set to the 600 ml bag is then heat sealed.The 600 ml bag is then removed and discarded.

Fifth, the regenerative cell composition is washed. A metal clip isplaced on the tubing between the two full bags to seal the tubing. Thequad set is placed on a balance. Water is added to a second “dummy” quadset to balance the quad set. The quad set and balanced set are placed onopposite buckets of the centrifuge. For the hollow filter, the cells arewashed and placed in the bag, and tubing is sealed between the bag andthe hollow fiber filter assembly described above. Using a peristalticpump, the fluid is run through the filter assembly and the cellconcentrate is collected in a bag on the downstream end. Care is takento make sure the quad set bags are not compressed and are upright. Thecentrifuge is operated at 400×g for 10 minutes. The quad set is removedfrom the centrifuge and placed in the plasma expressor. Care is taken toplace the bags in the expressor in such a way that the hard tubing atthe top of the bag is just at the top of the backplate. If the bag istoo high, too much saline will be retained, if it is too low the tubingwill interfere with the front plate's ability to close and again toomuch saline will be retained. A blue clamp is applied to each of thelines leading from the full quad set to the empty one. The metal loopsand blue clamps are removed to allow supernatant to flow into the emptyquad set. As much saline as possible is expressed off, but care is takennot to dislodge the cell pellet. The tubing running into each of thebags containing supernatant is heat sealed. The waste bags containingthe supernatant are removed. Blue clamps are applied to the tubingleading to each of the quad set bags containing cells. The bags aretaken out of the plasma expressor. A sterile connecting device is usedto connect the tubing leading to the quad pack to the saline bag. Theblue clamp leading to one of the quad set bags is removed to allowapproximately 150 ml saline to flow into the bag, and then the clamp isreapplied to stop the flow of saline. The full quad set bag is theninverted approximately 10-15 times for approximately 15 seconds. Theblue clamp leading to the empty quad set bag is then removed and all ofthe contents of full bag are drained into the empty bag. The metal loopclamp is reapplied to seal the tubing between two quad set bags. Thetubing is then heat sealed and the saline bag is removed. The full quadset bag is then inverted approximately 10-15 times over approximately 15seconds. Another dummy quad set is placed on a balance and isre-balanced to the cell quad set. The quad set bags (one full, oneempty) are then placed into the centrifuge so that the quad set bags arenot compressed and are upright.

The centrifuge is operated at about 400×g for 10 minutes. The quad setis then removed from the centrifuge and is placed carefully in theplasma expressor in such a way that the hard tubing at the top of thebag is just at the top of the backplate. If the bag is too high too muchsaline will be retained, if it is too low the tubing will interfere withthe front plate's ability to close and again too much saline will beretained. The metal loop is removed to express all the supernatant fromthe full bag into the empty bag taking care not to dislodge theregenerative cell pellet. The tubing between the bags is sealed, and thefull (waste) bag is removed and discarded. A new sampling site coupleris then inserted into the remaining bag. The cells of the cell pelletare then resuspended in the residual saline (if any) to obtain aconcentration of regenerative cells. The resuspension can be performedby gentle manipulation of the bag (e.g., squeezing and rubbing).

A particular example of the system embodying the present invention isshown in FIG. 4. FIG. 4 illustrates an automated system and method forseparating and concentrating regenerative cells from tissue, e.g.,adipose tissue, suitable for re-infusion within a patient. In certainembodiments of the system shown in FIG. 4, the system further includesan automated step for aspirating a given amount of tissue from thepatient. The system shown in FIG. 4 is comprised of the disposable setshown in FIG. 13 which is connected to the re-usable component of thesystem shown in FIG. 14 to arrive at an automated embodiment of thesystem shown in FIG. 15A. The disposable set is connected to there-usable component through, e.g., an interlocking or docking device orconfiguration, which connects the disposable set to the re-usablecomponent such that the disposable set is securely attached to andassociated with the re-usable hardware component in a manner that theprocessing device present on the re-usable component can control andinterface with, i.e., send and receive signals to and from the variouscomponents of the disposable set as well as various components of there-usable component and other associated devices and systems.

The user may connect the disposable set to the re-usable component,input certain parameters using the user interface, e.g., the volume oftissue being collected, attach the system to the patient, and the systemautomatically performs all of the steps shown in FIG. 4 in anuninterrupted sequence using pre-programmed and/or user inputparameters. One such sequence is illustrated in FIG. 15B. Alternatively,the tissue may be manually aspirated from the patient by the user andtransported to system for processing, i.e., separation and concentrationof regenerative cells.

Specifically, as shown in FIG. 4, tissue, e.g., adipose tissue, may bewithdrawn from the patient using conduit 12 and introduced intocollection chamber 20. A detailed illustration of the collection chamberof FIG. 4 is shown in FIG. 5. As illustrated in FIG. 5, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula. The user may enter the estimatedvolume of tissue directed to the collection chamber 20 at this point.The tissue is introduced into the collection chamber 20 through an inletport 21 which is part of a closed fluid pathway that allows the tissue,saline and other agents to be added to the tissue in an aseptic manner.An optical sensor of the system, e.g., sensor 29, can detect when theuser input volume of tissue is present in the collection chamber 20. Incertain embodiments, if less tissue is present in the collection chamberthan the user input, the user will have the option to begin processingthe volume of tissue which is present in the collection chamber 20. Incertain embodiments, a portion of the tissue removed from the patientmay be directed to the sample chamber 60 through the use of a pump,e.g., a peristaltic pump, via a conduit, which may be activated via userinput utilizing the user interface.

A sensor 29 can signal the processing device present in the re-usablecomponent to activate the steps needed to wash and disaggregate thetissue. For example, the processing device may introduce a pre-setvolume of washing agent based on the volume of tissue collected usingautomated valves and pumps. This cycle may be repeated in the collectionchamber until the optical sensor determines that the effluent liquid issufficiently clear and devoid of unwanted material. For example, anoptical sensor 29 along the conduit leading out of the collectionchamber 12 b or 12 d can detect that the unwanted materials have beenremoved and can signal the processing device to close the requiredvalves and initiate the next step.

Next, the processing device may introduce a pre-programmed amount ofdisaggregation agent based on the volume of tissue collected. Theprocessing device may also activate agitation of the tissue in thecollection chamber for a preset period of time based on the initialvolume of tissue collected or based on user input. In the embodimentshown in FIG. 4, once the disaggregation agent, e.g., collagenase, isadded to the collection chamber 20 through the collagenase source 24,the motor in the collection chamber 20 is activated via the processingdevice. The motor activates the rotatable shaft 25 which is comprised ofa magnetic stirrer and a paddle-like device wherein one or more paddles25 a are rigidly attached to the filter cage 27 of a filter prefixed tothe collection chamber 28. The paddles agitate the in the presence ofthe disaggregation agent such that the regenerative cells separate fromthe tissue.

The solution in the collection chamber 20 is allowed to settle for apreset period of time. The buoyant portion of the solution is allowed torise to the top of the solution. Once the preset period of time elapses,the necessary valves and pumps are activated by the processing device toremove the non-buoyant portion to the waste chamber 40. The transferinto the waste chamber 40 continues until a sensor 29 along the conduitleading out of the collection chamber 12 b or 12 d can detect that thebuoyant fraction of the solution is about to be transferred to the wastechamber 30. For example, a sensor 29 along the conduit leading out ofthe collection chamber 12 b or 12 d can detect that the unwantedmaterials have been removed and can signal the processing device toclose the required valves.

At this time the non-buoyant fraction of the solution, i.e., theregenerative cell composition, is moved to the processing chamber 30.This is accomplished through the use of the necessary valves andperistaltic pumps. In certain embodiments, before transfer of theregenerative cell composition to the processing chamber 30, anadditional volume of saline may be added to the buoyant fraction ofsolution remaining in the collection chamber 20. Another wash cycle maybe repeated. After this cycle, the solution is allowed to settle and thenon-buoyant fraction (which contains the regenerative cells) istransported to the processing chamber 30 and the buoyant fraction isdrained to the waste chamber 40. The additional wash cycle is used tooptimize transfer of all the separated regenerative cells to theprocessing chamber 30.

Once the regenerative cell composition is transported to the processingchamber 30 by way of conduits 12, the composition may be subject to oneor more additional washing steps prior to the start of the concentrationphase. This ensures removal of waste and residual contaminants from thecollection chamber 20. Similarly, subsequent to the concentration step,the regenerative cell composition may be subjected to one or moreadditional washing steps to remove residual contaminants. The unwantedmaterials may be removed from the processing chamber 30 to the wastechamber 40 in the same manner, i.e., control of valves and pumps viasignals from the processing device, as described above.

The various embodiments of the processing chamber 30 shown in FIG. 4 aredescribed in detail below. The processing chamber 30 shown in FIG. 4 isin the form of a centrifuge chamber. A detailed illustration of theprocessing chamber of FIG. 4 is shown in FIGS. 7 and 8. Such aprocessing chamber 30 is generally comprised of a rotating seal network30.1 comprising an outer housing 30.2, one or more seals 30.3, one ormore bearings 30.4 and an attachment point 30.6 for connecting theprocessing chamber to the centrifuge device present in the re-usablecomponent of the system; one or more fluid paths 30.5 in the form ofconduits extending out from the rotating seal and ending in a centrifugechamber on each end which is in the form of an output chamber 50 housedin a frame 53 wherein the frame is comprised of one or more ports 52 andone or more handles to manually re-position the output chamber 50.

The rotating seal network 30.1 is included to ensure that the fluidpathways of the processing chamber can be maintained in a sterilecondition. In addition, the fluid pathways of the processing chamber canbe accessed in a sterile manner (e.g., to add agents or washingsolution) at any time, even while the centrifuge chamber of theprocessing chamber is spinning.

The rotating seal network 30.1 shown in FIGS. 7 and 8 includes arotating shaft comprised of two or more bearings 30.4, three or more lipseals 30.3, and an outer housing 30.2. In this embodiment, the bearings30.4 further comprise an outer and inner shaft (not shown) referred toherein as races. These races may be separated by precision groundspheres. The races and spheres comprising the bearings are preferablyfabricated with material suitable for contact with bodily fluid, or arecoated with material suitable for contact with bodily fluid. In apreferred embodiment, the races and spheres are fabricated using, forexample, silicone nitride or zirconia. Furthermore, in this embodiment,the three lip seals are comprised of a circular “U” shaped channel (notshown) as well as a circular spring (not shown). The circular “U” shapedchannel is preferably fabricated using flexible material such that aleakage proof junction with the rotating shaft of the rotating sealnetwork 30.1 is formed. Additionally, the lip seals are preferablyoriented in a manner such that pressure from the regenerative cellcomposition flowing through the processing chamber causes the sealassembly to tighten its junction with the rotating shaft by way ofincreased tension. The seals may be secured in position by way of one ormore circular clips (not shown) which are capable of expanding and/orcollapsing as needed in order to engage a groove in the outer housing30.2 of the rotating seal network 30.1. The heat generated by or nearthe rotating seal network 30.1 must be controlled to prevent lysis ofthe cells in the solution which is being moved through the passage. Thismay be accomplished by, for example, selecting a hard material forconstructing the rotating shaft, polishing the area of the rotatingshaft which comes in contact with the seals and minimizing contactbetween the rotating shaft and the seal.

In another embodiment the rotating seal network 30.1 is comprised of asingle rubber seal 30.3 and an air gasket (not shown). This seal andgasket provide a tortuous path for any biologic matter which couldcompromise the sterility of the system. In another embodiment therotating seal network 30.1 is comprised of multiple spring loaded seals30.3 which isolate the individual fluid paths. The seals 30.3 arefabricated of a material which can be sterilized as well as seal therotating shaft without lubricant. In another embodiment the rotatingseal network 30.1 is compromised of a pair of ceramic disks (not shown)which create the different fluid paths and can withstand the rotation ofthe system and not cause cell lysis. In another embodiment the fluidpathway is flexible and is allowed to wind and unwind with respect tothe processing chamber. This is accomplished by having the flexiblefluid pathway rotate one revolution for every two revolutions of theprocessing chamber 30. This eliminates the need for a rotating sealaltogether.

The regenerative cell composition is pumped from the collection chamber20 along a fluid path through the axis of rotation of the rotating sealnetwork 30.1 and then divides into a minimum of two fluid pathways 30.5each of which radiate outward from the central axis of the processingchamber 30 and terminate near the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (FIGS. 7 and 8). Accordingly, in a preferred embodiment, theprocessing chamber 30 is comprised of two or more output chambers 50 asshown in FIGS. 7 and 8. These output chambers 50 are positioned suchthat they are in one orientation during processing 30.7 and anotherorientation for retrieval of concentrated regenerative cells 30.8. Forexample, the output changes are tilted in one angle during processingand another angle for cell retrieval. The cell retrieval angle is morevertical than the processing angle. The two positions of the outputchamber 50 may be manually manipulated through a handle 53 whichprotrudes out of the processing chamber 30. The regenerative cells canbe manually retrieved from the output chambers 50 when they are in theretrieval orientation 30.8 using a syringe. In another embodiment, fluidpath 30.5 is constructed such that it splits outside the processingchamber and then connects to the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (not shown). In this embodiment, large volumes of regenerative cellcomposition and/or additives, solutions etc. may be transported to thecentrifuge chamber and/or the output chambers directly.

With reference to FIGS. 4 and 7-9, between the collection chamber 20 andthe processing chamber 30, a pump 34 and one or more valves 14 may beprovided. In a preferred embodiment, the valves 14 are electromechanicalvalves. In addition, sensors, such as pressure sensor 29, may beprovided in line with the processing chamber 30 and the collectionchamber 20. The valves, pumps and sensors act in concert with theprocessing device present on the re-usable component (FIG. 14) toautomate the concentration steps of the system.

The sensors detect the presence of the regenerative cell composition inthe centrifuge chambers and activate the centrifuge device throughcommunication with the processing device of the system. The regenerativecell composition is then subjected to a pre-programmed load for apre-programmed time based on the amount of tissue originally collectedand/or user input. In certain embodiments, this step may be repeatedeither automatically or through user input. For example, the compositionis subjected to a load of approximately 400 times the force of gravityfor a period of approximately 5 minutes. The output chamber 50 isconstructed such that the outer extremes of the chamber form a smallreservoir for the dense particles and cells. The output chamber 50retains the dense particles in what is termed a ‘cell pellet’, whileallowing the lighter supernatant to be removed through a fluid path,e.g., a fluid path which is along the axis of rotation of the rotatingseal network 30.1 and travels from the low point in the center of theprocessing chamber 30 through the rotating seal network 30.1 to thewaste container 40. The valves 14 and pumps 34 signal the processingdevice to activate steps to remove the supernatant to the wastecontainer 40 without disturbing the cell pellet present in the outputchamber 50.

The cell pellet that is obtained using the system shown in FIG. 4comprises the concentrated regenerative cells of the invention. In someembodiments, after the supernatant is removed and directed to the wastechamber 40, a fluid path 30.5 may be used to re-suspend the cell pelletthat is formed after centrifugation with additional solutions and/orother additives. Re-suspension of the cell pellet in this manner allowsfor further washing of the regenerative cells to remove unwantedproteins and chemical compounds as well as increasing the flow of oxygento the cells. The resulting suspension may be subjected to another loadof approximately 400 times the force of gravity for another period ofapproximately 5 minutes. After a second cell pellet is formed, and theresulting supernatant is removed to the waste chamber 40, a final washin the manner described above may be performed with saline or some otherappropriate buffer solution. This repeated washing can be performedmultiple times to enhance the purity of the regenerative cell solution.In certain embodiments, the saline can be added at any step as deemednecessary to enhance processing. The concentrations of regenerativecells obtained using the system shown in FIG. 4 may vary depending onamount of tissue collected, patient age, patient profile etc. Exemplaryyields are provided in Table 1.

The final pellet present in the output chamber 50 may then be retrievedin an aseptic manner using an appropriate syringe after the outputchamber 50 is positioned in the orientation appropriate for cellremoval. In other embodiments, the final pellet may be automaticallymoved to a container in the in the output chamber 50 which may beremoved and stored or used as needed. This container may be in anyappropriate form or size. For example, the container may be a syringe.In certain embodiments, the output container 50 itself may be heatsealed (either automatically or manually) and isolated from the othercomponents of the processing chamber for subsequent retrieval and use ofthe regenerative cells in therapeutic applications as described hereinincluding re-infusion into the patient. The cells may also be subject tofurther processing as described herein either prior to retrieval fromthe output chamber or after transfer to a second system or device. There-usable component shown in FIG. 14 is constructed such that it can beconnected to one or more additional systems or devices for furtherprocessing as needed.

As described herein, adipose tissue derived regenerative cells compriseseveral cell types that may be used to derive a therapeutic, structural,or cosmetic benefit within the general context of regenerative medicine.However, there are substantial practical issues associated with clinicaluse of cells obtained from a solid organ that are not addressed in theexisting art. Isolation of viable cells from adipose requires celldissociation with proteolytic enzymes, specifically those that targetextracellular matrix molecules (i.e., collagenase) within the tissues.Accordingly, these cells must be clinically safe before being used as ahuman or veterinary cell treatment.

Specifically, in accordance with an aspect of the present invention,prior to application of adipose tissue-derived cells into a recipientwith the intent to derive therapeutic, structural, or cosmetic benefit,the likelihood of eliciting an adverse event is minimized. In essence,the likelihood of harm is minimized in order to maximize the net benefitto the recipient. The issues associated with maximizing both safety andbenefit differ dependent upon the site into which the final cell productis placed, the route by which it is placed there, and factors that maybe specific to the recipient (for example, age, concomitant disorders,and concomitant medications). According to a feature of the presentinvention, and/or cell suspensions or supernatants obtained (e.g., afterthe final, post-digestion, saline wash of the ADCs recovered aftertissue digestion) are assayed for one or more of the clinically relevantparameters described herein.

Certain constants, however, must be addressed irrespective of theabove-mentioned variables. One such constant is the absence ofinfectious, toxic, or pyrogenic material or agents. To minimize thepotential for such unsafe components to be present within theregenerative cells, all non-biologic and biologic materials that contactthe cells and the adipose from which they were obtained should be eithersterile or aseptic during use. For example, such biologic andnon-biologic materials should contain negligible or no endotoxin, aproduct of gram negative bacteria which is not uncommonly present inmaterials of animal and human origin. In a particular example, allnon-biologic that contacts adipose tissue during and after the digestionprocedure are tested beforehand and/or declared pyrogen free by thesuppliers, as defined by containing <20.0 United States Pharmacopeia(USP) endotoxin units (EU)/device as prescribed in USP document USP 24;NF 19; and/or cell suspensions or supernatants obtained (e.g., after thefinal, post-digestion, saline wash of the ADCs recovered after tissuedigestion) are assayed for the clinically relevant parameters describedherein (e.g., sample supernatant can be assayed for endotoxin levels asdeclared acceptable for clinical use by the Center for Devices andRadiological Health (CDRH) of the FDA; <0.5 EU/ml. Endotoxin exposure inhumans may result in sepsis and subsequent multi-organ system failure.In addition to endotoxin, cells disaggregated from adipose withproteolytic enzymes should be evaluated for residual proteolyticactivity within the final cell preparation, since such activity inhumans or animals could result in undesired and untoward tissuedestruction.

There are, however, certain embodiment of the present invention in whichplacement of the active cell population along with components of theextracellular matrix present within the intact adipose would enhancebenefit. One such embodiment is in the setting of a cell-augmented fattransfer process performed to provide a cosmetic or structural benefit.In this setting the connective tissue matrix would provide a scaffoldupon which regenerative cells could grow and, over time, remodel toprovide benefit. For example, partial disaggregation may be performedwith one or more enzymes, which are removed from the at least a part ofthe adipose tissue early, relative to an amount of time that the enzymewould otherwise be left thereon to fully disaggregate the tissue. Such aprocess may require less processing time and would yield a product thatwould not be suitable for intravascular delivery but which may provesuperior to a more fully disaggregated product for certain applicationsusing local, non-vascular delivery. Examples of such applicationsinclude soft tissue filling in cosmetic or structural applications.

However, there are many other situations in which the presence ofresidual extracellular matrix material would cause significant problemsrather than benefit. For example, collagen is a classic stimulator ofplatelet aggregation. Initiation of this process within a blood vesselby intravascular delivery of collagen or large collagen fragments wouldresult in development of a thrombosis and/or thromboembolic event thatcould result in local tissue ischemia, which in the case of the braincould be evidenced by a stroke or in the heart by a heart attack. Thus,for intravascular delivery or in situations where accidental delivery ofmaterial to the vascular space could occur or even where the presence ofa collagenous matrix is unnecessary the effects of such cellpreparations on platelet aggregation should also be evaluated. Forinstance, concerning contaminants are the bits of collagen remainingafter tissue digestion, in addition to any soluble agonists in the cellsuspension. In accordance with one embodiment, any detectable level maybe clinically relevant or undesirable.

Similarly, residual adipocytes and free lipid within the adipose derivedregenerative cell preparation should be minimized for intravascular orsystemic delivery (intended or accidental), as systemic administrationof adipocytes or lipid may result in fat embolism, leading to possibleend organ ischemic damage as well as a pulmonary ventilation/perfusionmismatch.

Thus, the precise nature of the regenerative cells suitable for clinicalor veterinary use will be dependent upon a number of factors. However,generally speaking, the delivered cells will fall into one of twocategories; one for intravascular delivery, the second for non-systemicimplantation. Both embodiments are encompassed by the present invention.

In one embodiment, the systems and methods of the present invention asdescribed herein are comprised of sterile or aseptic non-biologic andbiologic components used in a closed or functionally closed fluid/tissuepathway such that exposure of tissue, cells, biologic, and non-biologicmaterials with contaminants, disaggregation agents is prevented orminimized, wherein the components contain negligible levels ofendotoxin, and further comprising a method of removing adipocytes andfree lipid from the regenerative cells such that the regenerative cellsare suitable for re-infusion into a patient. The adipocytes and freelipids may be removed from the regenerative cells by any of the methodsfor separating and concentrating regenerative cells described herein.

Although the foregoing systems and methods have been described hereinwith a degree of specificity, it is understood that the presentdisclosure is made by way of example only and that modifications to thestructure of the system and sequence of the methods specified may bemade by one of skill in the art and are intended to be encompassed bythe present invention.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties.

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure

EXAMPLES Example 1 Isolation of Clinically Safe Regenerative Cells fromHuman Adipose Tissue Materials and Methods: Regenerative CellPreparation

Human regenerative cells were collected and harvested by enzymaticdigestion of adipose tissue as follows:

Human adipose tissue was procured at an outpatient plastic surgeon'soffice by vacuum assisted or non-assisted liposuction from the patientinto a syringe using a blunt canula which was replaced with a sharpneedle (14 gauge) after liposuction. A 600 ml single blood-pack unitwith spike-currently using a needle as substitute, herein referred to as“tissue bag”, was prepared by opening the sampling site coupler andspike in the side port of the bag, then rendering it the sampling siteaseptic with an iodine wipe. The adipose tissue was then injected intothe bag via the sampling site, which was subsequently swabbed with aniodine wipe. This process was repeated until all desired tissue wascollected. The bag(s) were then transported to the MacroPore CellLaboratory, where the bag was positioned downstream and in line with abag of injectable saline that had been pre-warmed to 37° C. Tubing wasthen secured in line between the spike on the tissue bag and the salinebag, a hemostat clamp was placed on the tubing, and the clamp wasclosed. The spike, via the needle on the tissue bag, was then used toenter the saline bag through the rubber septum, which was pre-wiped withiodine prior to insertion of needle. The clamp was then released,allowing approximately 150 ml of saline to enter the tissue bag, afterwhich time the clamp was reclosed. The bag was then inverted 10-15 timeswithin approximately fifteen seconds. Tubing was then placed in linebetween the 3 L waste bag and the spike on the tissue bag. A secondclamp was applied to this tubing. The spike on the waste bag was thenused to enter the 600 ml bag, the tissue bag was hung in such a way asto invert it over the work surface, the tissue was allowed to settle forapproximately one minute by maintaining the bag statically. The clampwas then released and fluid was allowed to flow from the tissue bag tothe waste bag, then the tubing was clamped off just prior to the tissueentering the tubing. The tissue bag was then lowered from the worksurface. This entire rinsing process was then repeated depending uponhow many cycles is required to remove most of the red blood cells,detected visually by a red coloration of the tissue. A heat sealer wasthen used to seal the tubing between the waste and tissue bags at apoint approximately in the middle of the tubing. The waste bag was thenremoved and discarded and the sampling site rendered aseptic with aniodine wipe. The clamp between a fresh saline bag and the tissue bag wasthen opened to allow approximately 150 ml of saline to enter the tissuebag.

Working solution of Blendzyme 3® (Roche Diagnostics), was then preparedminutes to yield the following concentration of enzymes: Collagenase Iand II (0.5 Wunsch units/ml) and Thermolysin (241 Caseinase units/ml).This was achieved by removal of 1 ml of Blendzyme from the commercialvial via a 22 gauge needle attached to a syringe and dispensing thisinto a container previously filled with 300 ml of injectable saline. Theneedle was the replaced with a 0.2 μm filter and second 22G needle. Theworking solution was then incubated at 4° C. The enzyme working solutionwas then added to the tissue bag via the sampling site and the tissuebag was incubated in a rocking, 37° C. water bath for approximately 1hour (55±10 minutes). The bag was then hung, inverted over the worksurface, and allowed to sit for approximately three minutes. A clamp tothe closed tubing formerly leading to the waste bag was then attached tothe tubing. The 600 ml bag was inverted over the work surface and allowto sit for ˜3 minutes

Analyses: Safety Analyses

Isolated cells were then evaluated for their clinical safety bymeasuring endotoxin levels, soluble factors that induce plateletaggregation, residual proteolytic enzyme activity in human serum, andadipocyte frequency.

Endotoxin levels in the saline rinses and final ADC suspension wasmeasured at Infinity Laboratories (Littleton, Colo.) by their protocol #LOP-426. In brief, the assay was performed in a 96-well plate. The testsample or standard (0.1 ml) was added to a well. The plate was incubatedat 37±1° C. in a heating block for 10 minutes. Lysate (0.1 ml) was addedto each well and the reaction was initiated. The amount of endotoxin pertest was calculated from the standard curve. A positive product controlwas performed to show the test sample did not interfere with the lysatereaction. 0.50 EU/ml was added to the test sample. The pyrogen resultmust be within −50% to +200% (0.25-1.0 EU/ml) to show a lack ofinhibition or enhancement.

Proteolytic activity originating from the Blendzyme® solution used todigest the tissue was measured using fluorometric gelatinase andcaseinase assays, to detect Collagenase I and II and Thermolysinactivities, respectively.

Collagenase activity was measured using a commercial kit [EnzChekGelatinase/Collagenase Assay kit (cat# E-12055, Molecular Probes;Invitrogen Detection Systems, Eugene Oreg.)] In brief, gelatin wascombined with either sample supernatants obtained after the final,post-digestion, saline ADC wash, or serially diluted Blendzyme of knownconcentration (to achieve a standard curve) in wells of a 96-well plate.Assay samples were incubated for 24 hours in the dark, then meanfluorescence was measured (495 nm excitation and 515 nm emission), usinga Gemini XS Microplate reader. Collagenase activity in samples from ADCwashes were calculated based on the known collagenase concentrationsfrom the Blendzyme standard curve.

Thermolysin activity was measured using a commercial kit [EnzChekGelatinase/Collagenase Assay kit (cat# E-6638, Molecular Probes;Invitrogen Detection Systems, Eugene Oreg.)]. In brief, casein wascombined with either supernatants obtained after the final,post-digestion, saline wash of the ADCs, or serially diluted Blendzymeof known concentration (to achieve a standard curve) in wells of a96-well plate. Assay samples were incubated for 1 hour in the dark, thenmean fluorescence was measured (495 nm excitation and 515 nm emission),using a Gemini XS Microplate reader. Thermolysin activity in samplesfrom ADC washes were calculated based on the known Thermolysinconcentrations from the Blendzyme® standard curve.

Platelet aggregation by the final ADC preparation was determined using aclinically standard platelet aggregation protocol at the Scripps ClinicMedical Laboratories (La Jolla, Calif.). In brief, platelet rich plasma(prp) and platelet poor plasma (ppp) were obtained after bloodcollection from patients who had not taken aspirin or ibuprofen for 2weeks. PRP samples from each patient were adjusted by addition of PPP toobtain a final platelet count of between 250K to 350K/mm³. Adjusted PRPsamples were then combined with either supernatants obtained after thefinal, post-digestion, saline wash of the ADCs, or from known agonistsof platelet aggregation; either collagen, ADP, or Ristocetin incuvettes, and read in an aggregometer set at 37° C. for between 5 and 10minutes. Aggregation induced by soluble factors in the supernatants ofthe ADC samples was quantified by comparison with the known agonistinduced aggregation profiles.

Adipocytes were measured in the final output of adipose-derived cellafter removal of floating adipocytes and subsequent centrifugation andresuspension to further concentrate the cells. A 10 μl cell suspensionwas pipeted onto a pre-labeled glass slides. Using another glass slide,the 10 μl cell suspension was smeared or spread thinly across the glasssurface and then air-dried. The air-dried slides were fixed in 50%Acetone/0.015 M Sodium Citrate solution for 30 seconds, washed in tapwater for 10 seconds and then air-dried again. Slides were subsequentlystained with hematoxylin and eosin, using a standard staining protocol.After staining, the slides were analyzed on a bright-field microscope,adipocytes are recognized as 50-100 μm size cells with blue nucleus atthe side of the cells, other nucleated cells are recognized as 5-15 μmsize cells with blue nucleus in the middle of the cells. Percentadipocytes contamination was determined from an average of 5 randomfields from 6 separate canister processing.

ADC Surface Marker Characterization

Isolated cells were characterized by cell surface markers, as follows.Flow cytometric analyses were performed using a standardBecton-Dickinson FACSAria instrument equipped with a 488 nm solid statelaser and a 633 nm air cooled laser. The data were acquired and analyzedusing the FACSDiVa software. Non-cultured ADCs were stained withmonoclonal antibodies to CD31, CD34, CD45, CD151, CD9, CD184, ABCG2,CD133, CD146, CD105, CD36, CD13, CD29, CD71, CD106, CD104, CD117, CD49d,CD44, CD151, and CD90 and were analyzed for their fluorescence intensityas a function of cytoplasmic granularity (side scatter). Non-specificantibodies were used as negative controls. The frequency of the distinctpopulations is expressed as a percent of positive events occurring inthe nucleated cell region (R1) (defined in the forwards side scatter vsside scatter plot).

Results:

Cell suspensions or supernatants obtained after the final,post-digestion, saline wash of the ADCs recovered after tissue digestionwere assayed for the clinically relevant parameters described above.Sample supernatant contained 0.02% endotoxin, substantially lower thanis declared acceptable for clinical use by the Center for Devices andRadiological Health (CDRH) of the FDA; <0.5 EU/ml.

Collagenase and Thermolysin activity originating from the Blendzymesolution used to digest the adipose was measured in supernatants offinal cell preparations. These were assayed in human serum, since it isa main component that an ADC treatment would come in contact with inpatients. Additionally, human serum contains inhibitors of tissuespecific proteases, hence, their activity in human serum is the relevantassessment. Residual collagenase activity in the samples supernatantswas 99.9% inhibited by serum. Likewise, residual Thermolysin activitywas also 99.9% inhibited in serum. Sample supernatant Collagenaseactivity in human serum was 1.5×10⁻² units/ml, which, which presuming aminimal dilution of 4-5 orders of magnitude upon introduction intohumans, would be 1.5×10^(−6 or −7) units/ml. In comparison, we measuredendogenous Endogenous collagenase activities in human serum to be2.2×10⁻⁶ units/ml. Similarly, sample supernatant Thermolysin activity inserum was 1.7 units/ml, which, again, presuming a minimal dilution of4-5 orders of magnitude upon introduction into humans, would be1.7×10^(−4 or −5) units/ml. In comparison, we measured endogenousThermolysin activities in human serum to be 2.7×10⁻⁴ units/ml.

Adipocyte content ranged from between 0.01% to 0.04% (mean; 0.02±0.01%n=6) of total ADC cell suspensions. Finally, the percentage of plateletsthat aggregated in response to soluble factors within the supernatant ofADCs ranged from 0% to 1.67% (n=3), as compared to 83.7%±9.6, 74%±8.2,or 83.3±%±8.1 for collagen, ADP, and Restocetin, respectively.

Summary

The foregoing results indicate that the regenerative cells obtainedusing a manual embodiment of the systems and methods of the presentinvention are clinically safe. Accordingly, automated embodiments usingpre-sterilized components, and closed or functionally closedsterile/fluid tissue pathways would produce regenerative cells that areequally safe if not more safe than those produced by the manualembodiments.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An automated cell processing unit for isolating a concentratedpopulation comprising CD31 positive cells from adipose tissue obtainedfrom a subject, wherein said cell processing unit is configured tomaintain a closed system, comprising: a tissue collection chamber thatis configured to receive a first portion of unprocessed adipose tissuefrom said subject while maintaining a closed system, wherein said tissuecollection chamber comprises an aspiration port structured to be coupledto a suction device; a first filter that is disposed within said tissuecollection chamber, wherein said first filter is configured tosubstantially retain a first component of said unprocessed adiposetissue and substantially pass a second component of said unprocessedadipose tissue, such that said first filter substantially separates saidfirst component from said second component, and wherein said firstcomponent comprises a cell population that comprises adipose-derivedstem cells and said second component comprises lipid, blood, matureadipocytes and washing solution; a processing chamber that is joined tosaid tissue collection chamber by a conduit such that a closed system ismaintained, wherein said processing chamber comprises a second filter, acentrifuge, or a spinning membrane filter; a programmable processingdevice capable of communication with said tissue collection chamber andsaid processing chamber, wherein said processing device operates atleast one pump configured to use pressure to control ingress and egressof tissues through said automated adipose-derived stem cell processingunit; and an outlet wherein said outlet is configured to allow theaseptic removal of a concentrated population comprising CD31 positivecells obtained from said processing chamber.